Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of transporter proteins that are related to the chloride channel subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect ligand transport and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Transporters

[0002] Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0003] Transporters are generally classified by structure and the type of mode of action. In addition, transporters are sometimes classified by the molecule type that is transported, for example, sugar transporters, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of molecule (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters: Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 (1997) and http://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0004] The following general classification scheme is known in the art and is followed in the present discoveries.

[0005] Channel-type transporters. Transmembrane channel proteins of this class are ubiquitously found in the membranes of all types of organisms from bacteria to higher eukaryotes. Transport systems of this type catalyze facilitated diffusion (by an energy-independent process) by passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism. These channel proteins usually consist largely of a-helical spanners, although b-strands may also be present and may even comprise the channel. However, outer membrane porin-type channel proteins are excluded from this class and are instead included in class 9.

[0006] Carrier-type transporters. Transport systems are included in this class if they utilize a carrier-mediated process to catalyze uniport (a single species is transported by facilitated diffusion), antiport (two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy) and/or symport (two or more species are transported together in the same direction in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy).

[0007] Pyrophosphate bond hydrolysis-driven active transporters. Transport systems are included in this class if they hydrolyze pyrophosphate or the terminal pyrophosphate bond in ATP or another nucleoside triphosphate to drive the active uptake and/or extrusion of a solute or solutes. The transport protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated.

[0008] PEP-dependent, phosphoryl transfer-driven group translocators. Transport systems of the bacterial phosphoenolpyruvate:sugar phosphotransferase system are included in this class. The product of the reaction, derived from extracellular sugar, is a cytoplasmic sugar-phosphate.

[0009] Decarboxylation-driven active transporters. Transport systems that drive solute (e.g., ion) uptake or extrusion by decarboxylation of a cytoplasmic substrate are included in this class.

[0010] Oxidoreduction-driven active transporters. Transport systems that drive transport of a solute (e.g., an ion) energized by the flow of electrons from a reduced substrate to an oxidized substrate are included in this class.

[0011] Light-driven active transporters. Transport systems that utilize light energy to drive transport of a solute (e.g., an ion) are included in this class.

[0012] Mechanically-driven active transporters. Transport systems are included in this class if they drive movement of a cell or organelle by allowing the flow of ions (or other solutes) through the membrane down their electrochemical gradients.

[0013] Outer-membrane porins (of b-structure). These proteins form transmembrane pores or channels that usually allow the energy independent passage of solutes across a membrane. The transmembrane portions of these proteins consist exclusively of b-strands that form a b-barrel. These porin-type proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and eukaryotic plastids.

[0014] Methyltransferase-driven active transporters. A single characterized protein currently falls into this category, the Na+-transporting methyltetrahydromethanopterin:coenzyme M methyltransferase.

[0015] Non-ribosome-synthesized channel-forming peptides or peptide-like molecules. These molecules, usually chains of L- and D-amino acids as well as other small molecular building blocks such as lactate, form oligomeric transmembrane ion channels. Voltage may induce channel formation by promoting assembly of the transmembrane channel. These peptides are often made by bacteria and fungi as agents of biological warfare.

[0016] Non-Proteinaceous Transport Complexes. Ion conducting substances in biological membranes that do not consist of or are not derived from proteins or peptides fall into this category.

[0017] Functionally characterized transporters for which sequence data are lacking. Transporters of particular physiological significance will be included in this category even though a family assignment cannot be made.

[0018] Putative transporters in which no family member is an established transporter. Putative transport protein families are grouped under this number and will either be classified elsewhere when the transport function of a member becomes established, or will be eliminated from the TC classification system if the proposed transport function is disproven. These families include a member or members for which a transport function has been suggested, but evidence for such a function is not yet compelling.

[0019] Auxiliary transport proteins. Proteins that in some way facilitate transport across one or more biological membranes but do not themselves participate directly in transport are included in this class. These proteins always function in conjunction with one or more transport proteins. They may provide a function connected with energy coupling to transport, play a structural role in complex formation or serve a regulatory function.

[0020] Transporters of unknown classification. Transport protein families of unknown classification are grouped under this number and will be classified elsewhere when the transport process and energy coupling mechanism are characterized. These families include at least one member for which a transport function has been established, but either the mode of transport or the energy coupling mechanism is not known.

Ion channels

[0021] An important type of transporter is the ion channel. Ion channels regulate many different cell proliferation, differentiation, and signaling processes by regulating the flow of ions into and out of cells. Ion channels are found in the plasma membranes of virtually every cell in eukaryotic organisms. Ion channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, ion channels, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0022] Ion channels are generally classified by structure and the type of mode of action. For example, extracellular ligand gated channels (ELGs) are comprised of five polypeptide subunits, with each subunit having 4 membrane spanning domains, and are activated by the binding of an extracellular ligand to the channel. In addition, channels are sometimes classified by the ion type that is transported, for example, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of ion (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters (1997). Receptor and ion channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0023] There are many types of ion channels based on structure. For example, many ion channels fall within one of the following groups: extracellular ligand-gated channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying channels (INR), intercellular (gap junction) channels, and voltage gated channels (VIC). There are additionally recognized other channel families based on ion-type transported, cellular location and drug sensitivity. Detailed information on each of these, their activity, ligand type, ion type, disease association, drugability, and other information pertinent to the present invention, is well known in the art.

[0024] Extracellular ligand-gated channels, ELGs, are generally comprised of five polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J. Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem. 239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), Trends Pharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J. Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions: this serves as a means of identifying other members of the ELG family of proteins. ELG bind a ligand and in response modulate the flow of ions. Examples of ELG include most members of the neurotransmitter-receptor family of proteins, e.g., GABAI receptors. Other members of this family of ion channels include glycine receptors, ryandyne receptors, and ligand gated calcium channels.

The Voltage-gated Ion Channel (VIC) Superfamily

[0025] Proteins of the VIC family are ion-selective channel proteins found in a wide range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolution and diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993), Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci., Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. and W. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo- or heterooligomeric structures with several dissimilar subunits (e.g., al-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), but the channel and the primary receptor is usually associated with the a (or al) subunit. Functionally characterized members are specific for K⁺, Na⁺ or Ca⁺. The K⁺ channels usually consist of homotetrameric structures with each a-subunit possessing six transmembrane spanners (TMSs). The al and a subunits of the Ca²⁺ and Na⁺ channels, respectively, are about four times as large and possess 4 units, each with 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs. These large channel proteins form heterotetra-unit structures equivalent to the homotetrameric structures of most K⁺ the homotetrameric K⁺channels. Ion flux via the eukaryotic channels is generally controlled by the transmembrane electrical potential (hence the designation, voltage-sensitive) although some are controlled by ligand or receptor binding.

[0026] Several putative K⁺-selective channel proteins of the VIC family have been identified in prokaryotes. The structure of one of them, the KcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Å resolution. The protein possesses four identical subunits, each with two transmembrane helices, arranged in the shape of an inverted teepee or cone. The cone cradles the “selectivity filter” P domain in its outer end. The narrow selectivity filter is only 12 Å long, whereas the remainder of the channel is wider and lined with hydrophobic residues. A large water-filled cavity and helix dipoles stabilize K⁺ in the pore. The selectivity filter has two bound K⁺ ions about 7.5 Å apart from each other. Ion conduction is proposed to result from a balance of electrostatic attractive and repulsive forces.

[0027] In eukaryotes, each VIC family channel type has several subtypes based on pharmacological and electrophysiological data. Thus, there are five types of Ca²⁺ channels (L, N, P, Q and T). There are at least ten types of K⁺ channels, each responding in different ways to different stimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive [BK_(Ca), IK_(Ca), and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)]. There are at least six types of Na⁺ channels (I, II, III, μl, HI and PN3). Tetrameric channels from both prokaryotic and eukaryotic organisms are known in which each a-subunit possesses 2 TMSs rather than 6, and these two TMSs are homologous to TMSs 5 and 6 of the six TMS unit found in the voltage-sensitive channel proteins. KcsA of S. lividans is an example of such a 2 TMS channel protein. These channels may include the K_(Na) (Na⁺-activated) and K_(Vol) (cell volume-sensitive) K⁺ channels, as well as distantly related channels such as the Tokl K⁺ channel of yeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and the TREK-1 K⁺ channel of the mouse. Because of insufficient sequence similarity with proteins of the VIC family, inward rectifier K⁺ IRK channels (ATP-regulated; G-protein-activated) which possess a P domain and two flanking TMSs are placed in a distinct family. However, substantial sequence similarity in the P region suggests that they are homologous. The b, g and d subunits of VIC family members, when present, frequently play regulatory roles in channel activation/deactivation.

The Chloride Channel (CIC) Family

[0028] The CIC family is a large family consisting of dozens of sequenced proteins derived from Gram-negative and Gram-positive bacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer, K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M. -E., et al., (1994), J. Mol. Biol. 242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher, W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998), Annu. Rev. Physiol. 60: 689-717). These proteins are essentially ubiquitous, although they are not encoded within genomes of Haemophilus influenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae.Sequenced proteins vary in size from 395 amino acyl residues (M jannaschii) to 988 residues (man). Several organisms contain multiple CIC family paralogues. For example, Synechocystis has two paralogues, one of 451 residues in length and the other of 899 residues. Arabidopsis thaliana has at least four sequenced paralogues, (775-792 residues), humans also have at least five paralogues (820-988 residues), and C. elegans also has at least five (810-950 residues). There are nine known members in mammals, and mutations in three of the corresponding genes cause human diseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiae only have one ClC family member each. With the exception of the larger Synechocystis paralogue, all bacterial proteins are small (395-492 residues) while all eukaryotic proteins are larger (687-988 residues). These proteins exhibit 10-12 putative transmembrane a-helical spanners (TMSs) and appear to be present in the membrane as homodimers. While one member of the family, Torpedo ClC-O, has been reported to have two channels, one per subunit, others are believed to have just one.

[0029] All functionally characterized members of the CIC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions (cell volume regulation; membrane potential stabilization; signal transduction; transepithelial transport, etc.). Different homologues in humans exhibit differing anion selectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductance sequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5 channels and others exhibit outward rectifying currents with currents only at voltages more positive than +20 mV.

Chloride Channel Protein 3 (ClC3)

[0030] The novel human protein, and encoding gene, provided by the present invention is related to chloride channel proteins in general, and shows the highest degree of similarity to chloride channel protein 3 (ClC3) in particular. However, the human protein of the present invention has an alternatively splice amino (“N”) end compared with known human ClC3 proteins. Specifically, the protein of the present invention differs from the art-known human ClC3 protein provided in Genbank gi2599548, and is similar at the amino end to the ClC3 protein previously found in Xenopus laevis, which is provided in Genbank gi6634696. FIG. 2 provides an alignment of the amino sequence of the protein of the present invention against gi2599548, and an alignment of the amino end of the protein of the present invention against the amino end of gi6634696.

[0031] ClC3 shares significant sequence and structural similarities with all previously known members of the voltage-gated chloride channel family. ClC3 also shows a high degree of similarity to GEF1, which is an integral membrane protein found in Saccharomyces cerevisiae that plays important roles in respiration and iron-limited cell growth (Borsani et al., Genomics 1995 May 1;27(1):131-41).

[0032] Mutations in ion channel, such as chloride channels, are known to be associated with a wide variety of neurological and muscular disorders (Taine et al., Hum Genet 1998 February;102(2):178-81).

[0033] For a further review of chloride channels in general and ClC3 in particular, see Shimada et al., Am J Physiol Gastrointest Liver Physiol 2000 August;279(2):G268-76; Rae et al., Exp Eye Res 2000 March;70(3):339-48; Rae et al, Curr Eye Res 2000 February;20(2):144-52; Shepard et al, Am J Physiol 1999 Sepembwer;277(3 Pt 1):C412-24; Rae et al., Exp Eye Res 1998 March;66(3):347-59; and Rae et al, Curr Eye Res 1998 Mar.;17(3):264-75.

The Organellar Chloride Channel (O-CIC) Family

[0034] Proteins of the O-CIC family are voltage-sensitive chloride channels found in intracellular membranes but not the plasma membranes of animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268: 14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272: 12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272: 23880-23886).

[0035] They are found in human nuclear membranes, and the bovine protein targets to the microsomes, but not the plasma membrane, when expressed in Xenopus laevis oocytes. These proteins are thought to function in the regulation of the membrane potential and in transepithelial ion absorption and secretion in the kidney. They possess two putative transmembrane a-helical spanners (TMSs) with cytoplasmic N- and C-termini and a large luminal loop that may be glycosylated. The bovine protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The human nuclear protein is much smaller (241 residues). A C. elegans homologue is 260 residues long.

[0036] Transporter proteins, particularly members of the chloride channel subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

[0037] The present invention is based in part on the identification of amino acid sequences of human transporter peptides and proteins that are related to the chloride channel subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter activity in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis.

DESCRIPTION OF THE FIGURE SHEETS

[0038]FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the transporter protein of the present invention. (SEQ ID NO:1) In addition structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis.

[0039]FIG. 2 provides the predicted amino acid sequence of the transporter of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

[0040]FIG. 3 provides genomic sequences that span the gene encoding the transporter protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 27 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0041] The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a transporter protein or part of a transporter protein and are related to the chloride channel subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human transporter peptides and proteins that are related to the chloride channel subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these transporter peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the transporter of the present invention.

[0042] In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known transporter proteins of the chloride channel subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known chloride channel family or subfamily of transporter proteins.

Specific Embodiments Peptide Molecules

[0043] The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the transporter family of proteins and are related to the chloride channel subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIGS. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the transporter peptides of the present invention, transporter peptides, or peptides/proteins of the present invention.

[0044] The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprising the amino acid sequences of the transporter peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0045] As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0046] In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0047] The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the transporter peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0048] The isolated transporter peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. For example, a nucleic acid molecule encoding the transporter peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0049] Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0050] The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0051] The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the transporter peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

[0052] The transporter peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a transporter peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the transporter peptide. “Operatively linked” indicates that the transporter peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the transporter peptide.

[0053] In some uses, the fusion protein does not affect the activity of the transporter peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant transporter peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0054] A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A transporter peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the transporter peptide.

[0055] As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0056] Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the transporter peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0057] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0058] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0059] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J.

[0060] Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0061] Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the transporter peptides of the present invention as well as being encoded by the same genetic locus as the transporter peptide provided herein. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0062] Allelic variants of a transporter peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by the same genetic locus as the transporter peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0063]FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 27 different nucleotide positions. Some of these SNPs, particularly the SNPs located 5′ of the ORF and in the first intron, may affect control/regulatory elements.

[0064] Paralogs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0065] Orthologs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0066] Non-naturally occurring variants of the transporter peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the transporter peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a transporter peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0067] Variant transporter peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to transport ligand, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0068] Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0069] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as transporter activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0070] The present invention further provides fragments of the transporter peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0071] As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a transporter peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the transporter peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the transporter peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0072] Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in transporter peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

[0073] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0074] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci 663:48-62 (1992)).

[0075] Accordingly, the transporter peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature transporter peptide is fused with another compound, such as a compound to increase the half-life of the transporter peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature transporter peptide, such as a leader or secretory sequence or a sequence for purification of the mature transporter peptide or a pro-protein sequence.

Protein/Peptide Uses

[0076] The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a transporter-effector protein interaction or transporter-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0077] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0078] The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, transporters isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis. A large percentage of pharmaceutical agents are being developed that modulate the activity of transporter proteins, particularly members of the chloride channel subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

[0079] The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to transporters that are related to members of the chloride channel subfamily. Such assays involve any of the known transporter functions or activities or properties useful for diagnosis and treatment of transporter-related conditions that are specific for the subfamily of transporters that the one of the present invention belongs to, particularly in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis. The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems ((Hodgson, Bio/technology, 1992, Sepember. 10(9);973-80). Cell-based systems can be native, i.e., cells that normally express the transporter, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the transporter protein.

[0080] The polypeptides can be used to identify compounds that modulate transporter activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the transporter. Both the transporters of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the transporter. These compounds can be further screened against a functional transporter to determine the effect of the compound on the transporter activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the transporter to a desired degree.

[0081] Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the transporter protein and a molecule that normally interacts with the transporter protein, e.g. a substrate or a component of the signal pathway that the transporter protein normally interacts (for example, another transporter). Such assays typically include the steps of combining the transporter protein with a candidate compound under conditions that allow the transporter protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the transporter protein and the target, such as any of the associated effects of signal transduction such as changes in membrane potential, protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

[0082] Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0083] One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant transporters or appropriate fragments containing mutations that affect transporter function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

[0084] The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) transporter activity. The assays typically involve an assay of events in the signal transduction pathway that indicate transporter activity. Thus, the transport of a ligand, change in cell membrane potential, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the transporter protein dependent signal cascade can be assayed.

[0085] Any of the biological or biochemical functions mediated by the transporter can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the transporter can be assayed. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis.

[0086] Binding and/or activating compounds can also be screened by using chimeric transporter proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a ligand-binding region can be used that interacts with a different ligand then that which is recognized by the native transporter. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the transporter is derived.

[0087] The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the transporter (e.g. binding partners and/or ligands). Thus, a compound is exposed to a transporter polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble transporter polypeptide is also added to the mixture. If the test compound interacts with the soluble transporter polypeptide, it decreases the amount of complex formed or activity from the transporter target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the transporter. Thus, the soluble polypeptide that competes with the target transporter region is designed to contain peptide sequences corresponding to the region of interest.

[0088] To perform cell free drug screening assays, it is sometimes desirable to immobilize either the transporter protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0089] Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of transporter-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a transporter-binding protein and a candidate compound are incubated in the transporter protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the transporter protein target molecule, or which are reactive with transporter protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0090] Agents that modulate one of the transporters of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0091] Modulators of transporter protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the transporter pathway, by treating cells or tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. These methods of treatment include the steps of administering a modulator of transporter activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0092] In yet another aspect of the invention, the transporter proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the transporter and are involved in transporter activity. Such transporter-binding proteins are also likely to be involved in the propagation of signals by the transporter proteins or transporter targets as, for example, downstream elements of a transporter-mediated signaling pathway. Alternatively, such transporter-binding proteins are likely to be transporter inhibitors.

[0093] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a transporter protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a transporter-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the transporter protein.

[0094] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a transporter-modulating agent, an antisense transporter nucleic acid molecule, a transporter-specific antibody, or a transporter-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0095] The transporter proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. The method involves contacting a biological sample with a compound capable of interacting with the transporter protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0096] One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0097] The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered transporter activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0098] In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0099] The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the transporter protein in which one or more of the transporter functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and transporter activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0100] The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. Accordingly, methods for treatment include the use of the transporter protein or fragments.

Antibodies

[0101] The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0102] As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0103] Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0104] In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0105] Antibodies are preferably prepared from regions or discrete fragments of the transporter proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or transporter/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0106] An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0107] Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

[0108] The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0109] Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0110] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0111] Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0112] The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0113] The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the transporter peptide to a binding partner such as a ligand or protein binding partner. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0114] The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

[0115] The present invention further provides isolated nucleic acid molecules that encode a transporter peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the transporter peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0116] As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0117] Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0118] For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0119] Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0120] The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0121] The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIGS. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0122] In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0123] The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0124] As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the transporter peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0125] Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0126] The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the transporter proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0127] The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

[0128] A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0129] A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0130] Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0131]FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 27 different nucleotide positions. Some of these SNPs, particularly the SNPs located 5′ of the ORF and in the first intron, may affect control/regulatory elements.

[0132] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1 989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

[0133] The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 27 different nucleotide positions.

[0134] The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0135] The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0136] The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0137] The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0138] The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0139] The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0140] The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0141] The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0142] The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0143] The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0144] The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis.

[0145] Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in transporter protein expression relative to normal results.

[0146] In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.

[0147] Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a transporter protein, such as by measuring a level of a transporter-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a transporter gene has been mutated. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis.

[0148] Nucleic acid expression assays are useful for drug screening to identify compounds that modulate transporter nucleic acid expression.

[0149] The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the transporter gene, particularly biological and pathological processes that are mediated by the transporter in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis. The method typically includes assaying the ability of the compound to modulate the expression of the transporter nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired transporter nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the transporter nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0150] The assay for transporter nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the transporter protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0151] Thus, modulators of transporter gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of transporter mRNA in the presence of the candidate compound is compared to the level of expression of transporter mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0152] The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate transporter nucleic acid expression in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0153] Alternatively, a modulator for transporter nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the transporter nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis.

[0154] The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the transporter gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0155] The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in transporter nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in transporter genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the transporter gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the transporter gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a transporter protein.

[0156] Individuals carrying mutations in the transporter gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 27 different nucleotide positions. Some of these SNPs, particularly the SNPs located 5′ of the ORF and in the first intron, may affect control/regulatory elements. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 4 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0157] Alternatively, mutations in a transporter gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0158] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0159] Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant transporter gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al.,Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0160] Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0161] The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the transporter gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 27 different nucleotide positions. Some of these SNPs, particularly the SNPs located 5′ of the ORF and in the first intron, may affect control/regulatory elements.

[0162] Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0163] The nucleic acid molecules are thus useful as antisense constructs to control transporter gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of transporter protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into transporter protein.

[0164] Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of transporter nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired transporter nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the transporter protein, such as ligand binding.

[0165] The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in transporter gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired transporter protein to treat the individual.

[0166] The invention also encompasses kits for detecting the presence of a transporter nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in teratocarcinomas of neuronal precursor cells, embryos (particularly in the head), duodenal adenocarcinomas of the small intestine, small and large cell carcinomas of the lung, breast tissue, Schwannoma tumors, brain tumors, and testis, as indicated by virtual northern blot analysis. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting transporter nucleic acid in a biological sample; means for determining the amount of transporter nucleic acid in the sample; and means for comparing the amount of transporter nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect transporter protein mRNA or DNA.

Nucleic Acid Arrays

[0167] The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0168] As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[0169] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0170] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0171] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0172] In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0173] Using such arrays, the present invention provides methods to identify the expression of the transporter proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the transporter gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 27 different nucleotide positions. Some of these SNPs, particularly the SNPs located 5′ of the ORF and in the first intron, may affect control/regulatory elements.

[0174] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0175] The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0176] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0177] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0178] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified transporter gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host cells

[0179] The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0180] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0181] The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

[0182] Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0183] The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0184] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0185] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0186] A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0187] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0188] The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0189] The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0190] As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterotransporter. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0191] Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0192] The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kuijan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0193] The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0194] In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

[0195] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0196] The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0197] The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0198] The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0199] Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0200] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0201] Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0202] While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0203] Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as transporters, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0204] Where the peptide is not secreted into the medium, which is typically the case with transporters, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0205] It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

[0206] The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a transporter protein or peptide that can be further purified to produce desired amounts of transporter protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0207] Host cells are also useful for conducting cell-based assays involving the transporter protein or transporter protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native transporter protein is useful for assaying compounds that stimulate or inhibit transporter protein function.

[0208] Host cells are also useful for identifying transporter protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant transporter protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native transporter protein.

[0209] Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a transporter protein and identifying and evaluating modulators of transporter protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0210] A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the transporter protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0211] Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transporter protein to particular cells.

[0212] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0213] In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0214] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0215] Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, transporter protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo transporter protein function, including ligand interaction, the effect of specific mutant transporter proteins on transporter protein function and ligand interaction, and the effect of chimeric transporter proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more transporter protein functions.

[0216] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

1 6 1 3625 DNA Human 1 gaacccagtt gcttcagcga gtcgaactac agttttaacc tcatcaaata tggcatctcc 60 cttgcttgct gcagcaggga tggaagaaat gtcactttct ttttaagcta gcaagctttt 120 tctttttctt tttcttcttc tatttaaaaa ttctaatcat ggatgcttct tccgaccctt 180 atttgcctta tgacggggga ggagacaata ttcccctgag ggaattacat aaaagaggaa 240 ctcattatac aatgacaaat ggaggcagca ttaacagttc tacacattta ctggatcttt 300 tggatgaacc aattccaggt gttggtacat atgatgattt ccatactatt gattgggtgc 360 gagaaaaatg taaagacaga gaaaggcata gacggatcaa cagcaaaaag aaagaatcag 420 catgggaaat gacaaaaagt ttgtatgatg cgtggtcagg atggctagta gtaacactaa 480 caggattggc atcaggggca ctggccggat taatagacat tgctgccgat tggatgactg 540 acctaaagga gggcatttgc cttagtgcgt tgtggtacaa ccacgaacag tgctgttggg 600 gatctaatga aacaacattt gaagagaggg ataaatgtcc acagtggaaa acatgggcag 660 aattaatcat aggtcaagca gagggtcctg gttcttatat catgaactac ataatgtaca 720 tcttctgggc cttgagtttt gcctttcttg cagtttccct ggtaaaggta tttgctccat 780 atgcctgtgg ctctggaatt ccagagatta aaactatttt aagtggattc atcatcagag 840 gttacttggg aaaatggact ttaatgatta aaaccatcac attagtcctg gctgtggcat 900 caggtttgag tttaggaaaa gaaggtcccc tggtacatgt tgcctgttgc tgcggaaata 960 tcttttccta cctctttcca aagtatagca caaacgaagc taaaaaaagg gaggtgctat 1020 cagctgcctc agctgcaggg gtttctgtag cttttggtgc accaattgga ggagttcttt 1080 ttagcctgga agaggttagc tattattttc ctctcaaaac tttatggaga tcattttttg 1140 ctgctttagt ggctgcattt gttttgaggt ccatcaatcc atttggtaac agccgtctgg 1200 tcctttttta tgtggagtat catacaccat ggtacctttt tgaactgttt ccttttattc 1260 ttctaggggt atttggaggg ctttggggag cctttttcat tagggcaaat attgcctggt 1320 gtcgtcgacg caagtccacg aaatttggaa agtatcccgt tctggaagtc attattgttg 1380 cagccattac tgctgtgata gccttcccta atccatacac taggctaaac accagtgaac 1440 tgatcaaaga gctttttaca gactgtggtc ccctggaatc ctcttctctt tgtgactaca 1500 gaaatgacat gaatgccagt aaaattgtcg atgacattcc tgatcgtcca gcaggcattg 1560 gagtatattc agctatatgg cagttatgcc tggcactcat atttaaaatc ataatgacag 1620 tattcacttt tggcatcaag gttccatcag gcttgttcat ccccagcatg gccattggag 1680 cgatcgcagg aaggattgtg gggattgcgg tggagcagct tgcctactat caccacgact 1740 ggtttatctt taaggagtgg tgtgaggtcg gggctgattg cattacacct ggcctttatg 1800 ccatggttgg tgctgctgca tgcttaggtg gtgtgacaag aatgactgtc tccctggtgg 1860 ttattgtttt tgagcttact ggaggcttgg aatatattgt tccccttatg gctgcagtca 1920 tgaccagtaa atgggttgga gatgcctttg gcagggaagg catttatgaa gcacacatcc 1980 gattaaatgg ataccctttc ttggatgcaa aagaagaatt cactcatacc accctggctg 2040 ctgacgttat gagacctcga aggaatgatc ctcccttagc tgtcctgaca caggacaata 2100 tgacagtgga tgatatagaa aacatgatta atgaaaccag ctacaatgga tttcctgtca 2160 taatgtcaaa agaatctcag agattagtgg gatttgccct cagaagagac ctgacaattg 2220 caatagaaag tgccaggaaa aaacaagaag gtatcgttgg cagttctcgg gtgtgttttg 2280 cacagcacac cccatctctt ccagcagaaa gtcctcggcc attgaagctt cgaagcattc 2340 ttgacatgag cccttttaca gtgacagacc acaccccaat ggagattgtg gtggatattt 2400 tccgaaagct gggactgagg cagtgccttg taactcacaa tgggcgcctc cttggcatta 2460 taacaaaaaa agatatcctc cggcatatgg cccagacggc aaaccaagac cccgcttcaa 2520 taatgttcaa ctgaatctca cagatgagga gagagaagaa acggaagagg aagtttattt 2580 gttgaatagc acaactcttt aacctgaggg agtcatctac ttttttttcc tcctttacaa 2640 aaaaagaaag gaaatataaa agccgggttt ttgcaacatg gtttgcaaat aatgctggtg 2700 gaatggagga gttgtttggg gagggaaagg agagagaagg aaaggagtga ggtatttccc 2760 gtctaacaga aagcagcgta tcaactccta ttgttctgca ctggatgcat tcagctgagg 2820 atgtgcctga tagtgcaggc ttgcgcctca acagagatga cagcagagtc ctcgagcacc 2880 tggcctgttg ctccaacatt gcaaagacac attatcagtc cctatttcta gagggattac 2940 tttgaattga gccatctata aaactgcaag gtcttgccct tttttttaat caaaactgtt 3000 ctgtttaatt catgaattgt atagttaagc attacctttc tacattccag aagagccttt 3060 atttctctct ctctctctct ctctctctct ctctctactg agctgtaaca aagcctcttt 3120 aaatcggtgt atccttttga agcagtcctt tctcatattg agatgtactg tgattttact 3180 gaggtttcat cacaagaagg gagtgtttct tgtgccatta accatgtagt ttgtaccatc 3240 actaaatgct tggaacagta cacatgcacc acaacaaagg ctcatcaaac aggtaaagtc 3300 tcgaaggaag cgagaacgaa atctctcatt gtgtgccgtg tggctcaaaa ccgaaaacaa 3360 tgaagcttgg ttttaaagga taaagttttc ttttttgttt tcctctcaga ctttatggat 3420 aatgtgaccg ggtcttatgc aaattttcta tttctaaaac tactactatg atatacaagt 3480 gctgttgagc ataattaaat aaaatgctgc tgctttgaca gtaaagagaa aaaaaaaaaa 3540 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600 aaaaaaaaaa aaaaaaaaaa aaaaa 3625 2 791 PRT Human 2 Met Asp Ala Ser Ser Asp Pro Tyr Leu Pro Tyr Asp Gly Gly Gly Asp 1 5 10 15 Asn Ile Pro Leu Arg Glu Leu His Lys Arg Gly Thr His Tyr Thr Met 20 25 30 Thr Asn Gly Gly Ser Ile Asn Ser Ser Thr His Leu Leu Asp Leu Leu 35 40 45 Asp Glu Pro Ile Pro Gly Val Gly Thr Tyr Asp Asp Phe His Thr Ile 50 55 60 Asp Trp Val Arg Glu Lys Cys Lys Asp Arg Glu Arg His Arg Arg Ile 65 70 75 80 Asn Ser Lys Lys Lys Glu Ser Ala Trp Glu Met Thr Lys Ser Leu Tyr 85 90 95 Asp Ala Trp Ser Gly Trp Leu Val Val Thr Leu Thr Gly Leu Ala Ser 100 105 110 Gly Ala Leu Ala Gly Leu Ile Asp Ile Ala Ala Asp Trp Met Thr Asp 115 120 125 Leu Lys Glu Gly Ile Cys Leu Ser Ala Leu Trp Tyr Asn His Glu Gln 130 135 140 Cys Cys Trp Gly Ser Asn Glu Thr Thr Phe Glu Glu Arg Asp Lys Cys 145 150 155 160 Pro Gln Trp Lys Thr Trp Ala Glu Leu Ile Ile Gly Gln Ala Glu Gly 165 170 175 Pro Gly Ser Tyr Ile Met Asn Tyr Ile Met Tyr Ile Phe Trp Ala Leu 180 185 190 Ser Phe Ala Phe Leu Ala Val Ser Leu Val Lys Val Phe Ala Pro Tyr 195 200 205 Ala Cys Gly Ser Gly Ile Pro Glu Ile Lys Thr Ile Leu Ser Gly Phe 210 215 220 Ile Ile Arg Gly Tyr Leu Gly Lys Trp Thr Leu Met Ile Lys Thr Ile 225 230 235 240 Thr Leu Val Leu Ala Val Ala Ser Gly Leu Ser Leu Gly Lys Glu Gly 245 250 255 Pro Leu Val His Val Ala Cys Cys Cys Gly Asn Ile Phe Ser Tyr Leu 260 265 270 Phe Pro Lys Tyr Ser Thr Asn Glu Ala Lys Lys Arg Glu Val Leu Ser 275 280 285 Ala Ala Ser Ala Ala Gly Val Ser Val Ala Phe Gly Ala Pro Ile Gly 290 295 300 Gly Val Leu Phe Ser Leu Glu Glu Val Ser Tyr Tyr Phe Pro Leu Lys 305 310 315 320 Thr Leu Trp Arg Ser Phe Phe Ala Ala Leu Val Ala Ala Phe Val Leu 325 330 335 Arg Ser Ile Asn Pro Phe Gly Asn Ser Arg Leu Val Leu Phe Tyr Val 340 345 350 Glu Tyr His Thr Pro Trp Tyr Leu Phe Glu Leu Phe Pro Phe Ile Leu 355 360 365 Leu Gly Val Phe Gly Gly Leu Trp Gly Ala Phe Phe Ile Arg Ala Asn 370 375 380 Ile Ala Trp Cys Arg Arg Arg Lys Ser Thr Lys Phe Gly Lys Tyr Pro 385 390 395 400 Val Leu Glu Val Ile Ile Val Ala Ala Ile Thr Ala Val Ile Ala Phe 405 410 415 Pro Asn Pro Tyr Thr Arg Leu Asn Thr Ser Glu Leu Ile Lys Glu Leu 420 425 430 Phe Thr Asp Cys Gly Pro Leu Glu Ser Ser Ser Leu Cys Asp Tyr Arg 435 440 445 Asn Asp Met Asn Ala Ser Lys Ile Val Asp Asp Ile Pro Asp Arg Pro 450 455 460 Ala Gly Ile Gly Val Tyr Ser Ala Ile Trp Gln Leu Cys Leu Ala Leu 465 470 475 480 Ile Phe Lys Ile Ile Met Thr Val Phe Thr Phe Gly Ile Lys Val Pro 485 490 495 Ser Gly Leu Phe Ile Pro Ser Met Ala Ile Gly Ala Ile Ala Gly Arg 500 505 510 Ile Val Gly Ile Ala Val Glu Gln Leu Ala Tyr Tyr His His Asp Trp 515 520 525 Phe Ile Phe Lys Glu Trp Cys Glu Val Gly Ala Asp Cys Ile Thr Pro 530 535 540 Gly Leu Tyr Ala Met Val Gly Ala Ala Ala Cys Leu Gly Gly Val Thr 545 550 555 560 Arg Met Thr Val Ser Leu Val Val Ile Val Phe Glu Leu Thr Gly Gly 565 570 575 Leu Glu Tyr Ile Val Pro Leu Met Ala Ala Val Met Thr Ser Lys Trp 580 585 590 Val Gly Asp Ala Phe Gly Arg Glu Gly Ile Tyr Glu Ala His Ile Arg 595 600 605 Leu Asn Gly Tyr Pro Phe Leu Asp Ala Lys Glu Glu Phe Thr His Thr 610 615 620 Thr Leu Ala Ala Asp Val Met Arg Pro Arg Arg Asn Asp Pro Pro Leu 625 630 635 640 Ala Val Leu Thr Gln Asp Asn Met Thr Val Asp Asp Ile Glu Asn Met 645 650 655 Ile Asn Glu Thr Ser Tyr Asn Gly Phe Pro Val Ile Met Ser Lys Glu 660 665 670 Ser Gln Arg Leu Val Gly Phe Ala Leu Arg Arg Asp Leu Thr Ile Ala 675 680 685 Ile Glu Ser Ala Arg Lys Lys Gln Glu Gly Ile Val Gly Ser Ser Arg 690 695 700 Val Cys Phe Ala Gln His Thr Pro Ser Leu Pro Ala Glu Ser Pro Arg 705 710 715 720 Pro Leu Lys Leu Arg Ser Ile Leu Asp Met Ser Pro Phe Thr Val Thr 725 730 735 Asp His Thr Pro Met Glu Ile Val Val Asp Ile Phe Arg Lys Leu Gly 740 745 750 Leu Arg Gln Cys Leu Val Thr His Asn Gly Arg Leu Leu Gly Ile Ile 755 760 765 Thr Lys Lys Asp Ile Leu Arg His Met Ala Gln Thr Ala Asn Gln Asp 770 775 780 Pro Ala Ser Ile Met Phe Asn 785 790 3 65359 DNA Human misc_feature (1)...(65359) n = A,T,C or G 3 aattctatac aaatataatt atatagatat atattacata tacacacaat tgtttatctt 60 taaaaataat tcaaatatgg ctacaaaact tttacaatat gaagcattgt cagtatttat 120 tttaccggga ggatttcccc catcagtgag tgctgactgt cattttcatt ctttatgatc 180 aagttgtaga tcaggaaaaa caagttaaga gagtgcctac aaataccggg aaaacttgtg 240 gatagatttt cattttttat gtaaagacat ataagaacat gaatggtata aaaacaaaat 300 cctttataaa tgccatacaa ttatatattt agaaaaatta tatggtggta aaacatataa 360 aagaaccaca cactcccaaa tttacattga gctaatttag tacagttagc ctttgtcaaa 420 gctttccttg tttaaaaaaa ctattggctc agtgtgcagg aaggagcata ggagaaaaaa 480 ttgccaagaa tatttgaaaa atacagaaaa taaagaaaaa aatcacctac tatcctatca 540 aaaattttaa tagctagaat caggataaga tagaatattc ctgtggcagt aattctagtc 600 tatattcctt tcctggaacc ctgtctccca aatttcaggt gagattttat aagaagctct 660 gtttatctga gatttaaaat ataaaaactt gatttaacct atacagtttt ttaaaaagac 720 cctaaataag taaaatttag tactccacaa attgaagaga atttctctct tctctttact 780 gccctctgag ttttctcttt ccttctctca cctccaattt tcatgtaaac actttcagtt 840 cgagtggacc ttagagattg tctcattcaa tactttagga aaacaaattt tatagaaccc 900 ttgagttctg tggaattgct tctaatgaac aacacctttt gttgttgttg ttgtttagtg 960 acactgtgta acaggcattt caggaggaga atctcccagt ctagaggaat cctctcagag 1020 gtagctataa aatattgaac tctgatcttc aataagcatt gtgcggtttt tgtttttgtt 1080 tttaatgaca gttttaaaca agaaagttgc tttatttctg aacttcataa aaatttctat 1140 taaagagaca atttctgaat tttataacaa tttctagaac agttgagtac ctcactttga 1200 gacacatttt tgctaaaagt taaaaacaca aaacccttat gagataaaat aggaagctag 1260 tagagatagg aaagtcctct gcttagtaaa cctctttttt gcgtagttta gacacataca 1320 atagtaaagt tacttagtac gttgatagtt ttctttctcc tcaaaagcta caatgtctta 1380 ctagctagtt ccttcaagaa aggaaacaag aagccgctgg aggagattgg tgagtgggat 1440 aaaacactat tcaactcttc agttattcgg tttttaaatc ctcaatgaaa ggctgctgta 1500 ttatagagta tttttttttt atttttaata gacttagaac caagtttctt gagaaacctt 1560 tggcatattg tagttttttt atggctatga ctcacatgac attactgtat aaaactagta 1620 cattctctcg taaaaccaca caaacttact agagtgctgc tctcattttt ctacattaga 1680 aatgaaaaag ggcattgtct gcattcaaaa tttccttttt acatctctgt attacttttt 1740 cccctttata tttatcttaa aaccaaaaga aataatgttt ctattgtttt actgtagtta 1800 ccactgatgc taccgaagct gtattgtgag tgtttcaaaa ttctcaaacc agttttgtgt 1860 gttgtacttg gagcttagtc attgtcatac gtagcaggac ctgattaaga aggctgtgcc 1920 gcctctaagc cttgctagat tgtagccact agcaaccagg ctgcaataat ttccctttga 1980 tgacatcatc cactgtggaa gaacccagtt gcttcagcga gtcgaactac agttttaacc 2040 tcatcaaata tggcatctcc cttgcttgct gcagcaggga tggaagaaat gtcactttct 2100 ttttaagcta gcaagctttt tctttttctt tttcttcttc tatttaaaaa ttctaatcat 2160 ggatgcttct tccgaccctt atttgcctta tgacggggga ggagacaata ttcccctgag 2220 ggaattacat aaaagaggta atactatccc cttgctgtga attctctgtt ggtatgtttt 2280 gcatgcggct gggcggtcct ctagcttaaa ctggttctcg tttgtccttt aaatactgca 2340 gtacgttgtt tagttgccct gggttgttag taaggggaaa atgcaacctt ctgaatggtt 2400 gtgtagccat ccctgattgt tttctctgtg cagattagta ctgcttcaga tcacgtcggg 2460 ctccgactcc atcttctgca tgaaaatctt ctttctaact ctgaaaatga attaatctgc 2520 ttttacagcc aactaaagtc gtgttggttg gcatctaaaa agtaatgttt ttcttccttc 2580 agaaaactta catttccttt aatttacaca gagaaatcag gtgcctatgt accattatat 2640 tttagctgct gccaattacc atgtagattt tacaccacaa agtaaattta tagcaaaagc 2700 tttacctaca ttttagaaca ttttaaaatg atagtaaaga tgaataattt ctatattaat 2760 actttttatt taatatgtat ttcggctgag taacatacta cattgtcttc cacaggtatc 2820 ttgtgaaatt tgatatgata aaacacattt gactaaatgt cagaaaaaat aatattggtt 2880 tgtgaaaagc agaagagcac ccagcatgcc tgtaaatctt ttggcaggca cttcctcagt 2940 ctccttaaaa ttaattgcat gttaattact accctttttt tcatttttgt ttaattgctt 3000 attcgaaaaa cagactggtc gacatttgtt gtcctagaaa aaaattgaac ttcaagaaaa 3060 atctcttagc ttatgtgact tcatttttga gccacattag tttgaattac tgcatgatat 3120 tataaactca ccttatgatt taacccaaac ttttatttgt aagtatataa ggaagtaata 3180 atgtttttct aatataatta gcctgcttta tttaaaatat actttgtgtt ctgataacac 3240 ttttttttta gtattaagtt ccactataat ttaaacatta taatgtattc aacaaatgtc 3300 tgttggttgc attgtgtctg ctacacacta ttttagggtc tgaacagttg tagcattatt 3360 tatcttgcag tattctgtag ttagtaaaaa cttgcttttt acattttgag aaaagctgtg 3420 taaggatcat gttacataca ttgtgctttc tcttacagag ttaccttctt aataaaattt 3480 tgatatatgt gtatatgtat atgttagaac atttggaaga aatatctaaa agcataaaga 3540 agaaaataat ttcttgtaat cacaccaccc agagcttttt aaattttttt tcttaatgtt 3600 acgatcataa attcttctat ttcctatgtt ctgattatca gttttctggt aaggagttct 3660 ttaaacagga agcaaggtga atgaatagtg actgttcaaa tgtcacatta tttgctaatc 3720 agtaattaaa ctgtaaaaca agacagactg tattttcctc atgctattac aacatttggt 3780 tgttaatgat gatagatcag aatacctggg cttcagaaat ttaaattcct tttgtgaagc 3840 ttaacagtct ttgacagaac ttacttatgg actgtcttag tgtaaaatat gcaaataata 3900 agaaataagt caaaacttat gtgagagtag gcatggttac tgatattacc taaacgtaag 3960 ctttttattt ctattatact ttcataaata atcctttaag aatcttgctt aggatctaaa 4020 tcagtcccac tcttggcagc tcaaataggt tctttatccc ttgatgagac ttattctatt 4080 aatataagtc attgttattt gaaagtaaca ttgtgtatgt gtagtagaga taagtcagtt 4140 attaggcttt cgtgactgta ctgtattacc tcaaacatac tgtagtatcc tagtgtctat 4200 gcgtaagatg ttattttttg tccataattt atgacctgtt gtagccatgg gtcaacacaa 4260 tggaattgat ggagacaggc agctaacaaa tcgaaaaaac tgaatcagct tccctgtgag 4320 gaagaacaaa actataatga ttaaaattga tcttcagcct gatagtgaag aggcagataa 4380 agtataaaat tgtgaaggat atcaataaag taaacatgga tctgtttagt aaatccctga 4440 gtgctatagc caaggattac ctttgttgag taaattgaat ttaatactac ttttcaaggc 4500 gagatggtaa atggtgaagc ttcctattta agtaaataat gtcaagtctg gaagtataag 4560 tagattcaaa ttagaattag tttgatatac tattgataga ttagaaatta agatgacatt 4620 tcagaaatag ccatctttag gggtagattt cctatataga aacaatcaag ctctctcaaa 4680 atgtctcttc cttttttatc aggaaaaaag acttggctta tctggactgt tagttttaca 4740 ctttttcttc ttaatttgtt caagatgttt aagtagtttt agaggtcaaa tttctttctt 4800 ctaccaaccc tttataatgg atttgattct tttgggcctg agcctccatt tactccatga 4860 ggggccttta acaattattt aaatnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5100 nnnnnnnnnn nnnnnaaaat agtaatatta ataatagtta atatttatta gaatttcctg 5160 ttagctggat actgtcccta agtgggtttt tttgttgttg ttgttgttgt tgttgttttc 5220 ttaagagaga ggtatcactt tttcacccag gctggagtgc agtggagtga ttatagcaaa 5280 tgcagccttg aactactggg cttagatcct ccgtctcacc ctccttggta cctgggactg 5340 caggcttgca acaccttgcc tggctaattt aaaaaacaaa attttttttt tttttaggga 5400 gagtctcact atgttgtcca ggctggtctc caactcctgg gctcaagcaa ccctcctgcc 5460 ttggcctccc aagtagctga gattacaggt gcgagccact gtgcctggct tgttctaagt 5520 gctttatgtg tatgaaatta tttaaatcct catcacaagt ttatgaagta ggtactgtta 5580 taatccccat tttctagttg acaagactga ggtaaggaat tgttaaggaa aagtcagaat 5640 tccatccaga tatttggctc atactttaat catgaggcta aactgcttct ctctacacgt 5700 atcttcatag taacttgtgt tttaagtctg gtagaagcat aagaagttta aacacagaca 5760 gaatcctgtg gaagttagta aatttctagt gaacgataga aatgatagaa atctcttctt 5820 cccccaaagt cccaagaaca gattagtctg cttttgacaa gtgttatcaa agtagactgt 5880 tctcacatac acgggggact caatagggca ttcctggtgg atataataaa atgagtaaat 5940 gcgataacag gaggaaatgc ctagtgtgtt gctcttggat tagttttgat acaacaaagg 6000 cagctttgtt gtgagtcagt agagagggta gtgtagaaag gtggaagttg gaagagtggc 6060 agatcctaga ggactaatga tgggcttaaa ccacaaaaag tgtcgctttg ccattgaaat 6120 aaaagtttgg ggtcttattt tttcaatttt ctccctgaaa ttatttcttg acattcatta 6180 gctcagcagt gtatctaaat aaagcttttt tgggtttcta ttataataga ggtttgttcc 6240 tttttcttcc ctttgaaaag tatcattttt tgcacattat ttgaaaatcc aggtgttata 6300 tgatattctt attgccagag ggacattctg caggctcttt gtaaaatgat tttaggattc 6360 agatacttat tatattttta ttggccctaa tattttatcc aactagaaaa ttaaacctct 6420 tcttaaaaat taatccatct aagtgtctgt aaattaaagg aacaactaaa gattctttat 6480 ttggtgtcag aaactccttg tttctacaac agtagtataa aacaaagcct gtttttaaat 6540 gtacttttcc cacagtatct gaatttcaaa tcttcaataa aatctggttc atattactac 6600 ctctagcttg attttctaaa aatagctgac actttagtat ggttaatttt atgccatctc 6660 atggcttgtc agaaatgctt tgtatcaaga tttccgagtg tgaacagatt tcctgccgca 6720 ttgattaagt ttgtaatttt ggctattttc ccagcatcga ggtttctgct ttgcgtttat 6780 gcaggagact ggtagtttaa attgaacttt aaggttttgt ttcttgtttt taagttaaca 6840 tatgtttaat ttctagtttc tttgtagccc tttgcaactt taattaggtc ataaaatgga 6900 tttactctag tttctctaac aaattttata aatttatgaa atatgaaatt tagcaaattt 6960 tataaacctt tttattcatg tattgtacag ctcatcatat ttgcagacat aataattgaa 7020 tgtggaactt gtttccaatt acacagatgt cttaatatcc accttatcat ctctaactaa 7080 aggatgtggc tttttatttt tgaggtggca acagaacaga aaagaaaaca gtgaattgag 7140 taatgggctt agtattgctg ctgcctggtt gtgtatcttt ggtaaacttc tttgagattt 7200 ggcattaact tgcaagtctt tgcagtttag acagttaaat atgactgaat ggctgaacaa 7260 attttaatag cgtatgcttc ttttttgcta tttatttacc cagtagacat ttaattgacc 7320 acctgctaaa tgtgaggcac tattcttgcc attacctttt taatctttga tttggagtct 7380 gctaacattc tggaacttcc actatcaact tagaacgttt actttcccat cccttaccag 7440 gatggccatt tcttatcagt agggtcacag agagagaaaa aaaaaaccat ctggggctag 7500 acttcctgct cttaacatac agaagcaaat aggttgtgaa ggaatacata gtattttgga 7560 tttctgcctc ttccttccat aattttttta aaaaggttca tatgttttat gtgtgtctta 7620 tgtaacagta atctgcatta tgaacttaaa tgacgaggat caccatttca catctttgga 7680 gattgatcac agaggtaata agtaactctt tttaaataac tatatgcatc atttttcatg 7740 taaaactatt atttggataa acccctttga gaaaaggctt aggctcctgc cagtgtcact 7800 gtgatattta ctaataagct cagtttaagg cgcagcaatt aaggttgtgt tgtttttttt 7860 tttttaagtt cagttcagca aatatatgtg gaaagcttgt gggtaaaatt atatttgtat 7920 ttttgggaaa gcagacaatt ttattaatgc ctatattttt ctagttcagt gtttgtcaaa 7980 cttcaagttt taacatgttg atcatgaaac cagttgactt gtgaccagta ttttaaaagg 8040 aaagattaaa aaaacaaaat aaaatatcag tatataccaa gtagtaagag taagcattgt 8100 ttactaaact ttggttttat ttaagtacat atctatatac tatgtcagtg agaaacattt 8160 ctccacttca tgtttgaaaa acatttcaaa agctaagaaa aagtttgaaa acctgtttgt 8220 aagtacacct ggggtaaagg tacaccctgt ggcataagat gtcgggaaca actgagggta 8280 agaatgggga tgcattacta tcgtaaactt ctgctaaagc ataaggatgt gagtgctggg 8340 agcaaagcag tgctcaccac ttctgcaatt ttctattgca gcattttaaa taatatggga 8400 aaaagtggac tgcaaccaaa ggcaaagagg gatggtgatg gtgaagggta agattgtatt 8460 tattgtccaa aggctaagtg catatacata tgtgtttggg agaaggcatc acgtaatagt 8520 tcttaaccta ctctgagaga aggttgtcca catttcttaa agtatacatg taaaccaaca 8580 atgaaattat tttagtgact tgagaatcaa agtgctagag tttgaatccc tgttctacta 8640 cttgctagcg gtgtgacctt gggcctgttt aactcttgac accttgtttt ccaaatttat 8700 aaagtggaga taataatatc tgtcacattg tgttgttgtg aggattatat gaactaatat 8760 atgtaatgtc ctgagaacaa tgtctggtac acattaagtt aattaaaatt agctgttctt 8820 actgttatta ttagacatga gctagataac agtggcctct acatgtgaaa gattatttta 8880 attctgatgt agttcagttt atctattttt tttatttttg tcccttttgc attgatgtca 8940 tatctaaaaa acctgcctaa ctcaggatca caaaaattta ctcctgtatt ttataatttt 9000 agctctttag atctaggatc catttttagc taatttttat atatggtgtg aggtaggggt 9060 acggtttcat tcttttgcac gtgaatagcc agttgtccca gcatcattta ttcaaaagac 9120 tattctttcc tcactagaaa aaatatttct ttaaagaata atgaatcctt ttttttttct 9180 ttttaaccgc tgttactcag ttggaaaaag aataatgaat aattttaagt aattttccta 9240 caggtaaatt taagtcttta tgtttagatt acacatatta ggaaataatg gatttgtatt 9300 ccataggtat gcttgatctt tataaagttc cctgtctctg gaaaaactaa aataaggcaa 9360 aacaatcttc ttagtagagt tatttttaca agaaagttgc aagccagttt tagttcatcg 9420 attggataat ttttcctgct tgctggaggt atttcagtat tggtaatacc tgaactatga 9480 ggatgcatga atgatgcatt ttaggaattt gtttctgtgt ccataccagg cataatgaat 9540 taagttatct gttaaaaata caggattttt gctcaatata cagttgtaga agaactcatt 9600 gtccaaattt ttaagacttt tttttctttt tttttttgag atggatctcg ctctgtcgcc 9660 caggttggag tgcagtggca caacctccac tcactgcaac ctccacctcc agggttcaag 9720 tgattctgct gcctcagctt cccgagtagc tggggactac aggcatatgc cactatgccc 9780 gcctgatttt ttttagtaga gatggggttt caccatattg gccaggctgc tcttgaactc 9840 ctgacctcgt gatccacccg cctcagcctc ccaaagttct gagattacag gtgtgagcca 9900 ccgcgcccgg ccagacattt tttttttttt tttttttttt gctgtctttg tcatattgtt 9960 agtcttttgg ttaagcgata ttataactta gtcatatgag taatataatg caacatgctg 10020 aattgtgtgt gtgagagggg gttgtttttt gtttgttatt tgttttttaa atagagatga 10080 gatctcactg tgtttcccag gctcccttga actcctgggc tcagatgata tagcctcctg 10140 ccacagcgtc ctgattagct gggactacag gtgtgcacca ctacacgtgg ctttcctgat 10200 gaaattttaa atacccaaat atttgagcag aaataatagc ttgtgtttat tgtttttcta 10260 ctatctgtca agtatagtat taaatgtttt acataatttg tctccagtcc acatacaata 10320 ctctagtaga agtgggtaac aaaaccaagg tactcaaaga ggttaataag taacttgcgc 10380 tggatcacag aactaacggg aggcagggct ggaatttgac tctaggtctt tctgacctca 10440 aagtgcagta aagtcatgga atttctctac taggccacct ggaagaaaag tgatcttttt 10500 tccagtcttt tttgttactg tttttcagcc aggagatagt agagttaggt agtagaatag 10560 tagtcactgg catccggtag tcagccctcc aaaaaagttt ttgatttttt tttttttttt 10620 tgtcttaaac ttggaagcta ctaactttca ggtcatactt tcttatcatc caagagctgg 10680 atatttaggt agcagaaact atggaattat cctaagtcct cttgaagctt cagctgttaa 10740 aattaattgg ttctgattaa cactgtgctc aagatttaca tttctaggag ccacagtttg 10800 attggtctaa cttggatcta tgtgttttct ttagctgggg aggagaaggt atcttgattg 10860 ataccttcac caggactgca tgcagtgagg gacagaagtt tccttaaaat aattgggttc 10920 tgttatagga agaaggggaa ggagatacca agtgggcaaa acaatcaggt tctattacat 10980 aaataataaa cctaatgtga cgataataaa tggataatat gattatttta agtttggaaa 11040 tatacctggt tattagtatt ggatatctgg tagtggggtt ggagaaaaag tcgagaataa 11100 gaaaagactt aaaatcgtaa aaattaactg gaaaagagga tggctgagca gatacatata 11160 tgttagataa tgttcataat ggcaaaccaa cctgaagatt tgtttaaatt gtagtatgta 11220 gccaggtgtg gtggtgcttg cctgcagtcc caactacttg ggaggctgag gcaggatgat 11280 tgcttgagcc taggtttgag gctacagtga gctatgtttc caccactgct ttccagccta 11340 ggtggcagag caagacccca tctctaaaaa aataaagtaa aatgaataaa ttataatatg 11400 ttatgacaaa tatagttatc tgaagtcaca gaaaatgtgc atgtgcattt aatgatgtga 11460 aataattttt aggaagtatg aataaaaaaa tcaactttta agtgtggcta gtatgatctt 11520 acctgtatct cacttataga aaatataaaa ggctgaagcc agtcaccagt ttaatagttc 11580 taacctcttg tttacttgat tccctttttt ctcctcccca gcaatcctca tatagttagg 11640 taaagttggt tcttcatcag gcttgttgca gaaaccccta agccttttta cttaaagctt 11700 tttgaaaccc agaaacccat cttttgaatt caaaagtttt gactgttatt agtctttttg 11760 tatgtttgtt ggccgcataa atgtctcctt tttatgaaca gagaagtgtc tgttaatata 11820 ctttgcccac tttttgatgg ggttgtttgt ttttttcttg tacatttgtt taagttcctt 11880 gtagattctg gatattagac ctatgtcaga tggatagatt gcaaaagttt tctcccattc 11940 tgtaggttgc ttgttcattc tgatgatagt ttcttttact gtgcagaagc tctttagttt 12000 aattagatcc tatttgtctg ttttggcttt tgtcgccatt gcttttggtg tttcagtcat 12060 gaagtctttg ccagtgccta tgtcctgaat ggtattgcct aggttttcat ggttttgggt 12120 tttacattta agcctcaaat cgatcttgag ttaatttttg tataaggtgt aaggaagggg 12180 tccagttcca gttttctgca tatggatagc cagttttccc agcaccattt attaatatta 12240 aatagggaat cctttcccca ttacttgttt ttgtcaagtt tgctgaagat cagatgattg 12300 tagatgtgtg gtgttatttc tgaggtcttt gttctgttcc gttggtctgt atatgtgttt 12360 tggtaccagt actatgctgt tttggttact gagccttgta gtatagtttg aagtcaggta 12420 gtatgatgcc tccagctttg ttatttttgc ttaggattgt cttggccata cgggctcttt 12480 tttggttcca tatgaaattt aaagtaggtt tttctaattt tgtgaggaaa gtcaatggta 12540 gcttgatggg aatagcgttg aatctataaa ttacttcggg cagtatggcc attttcatga 12600 tattgattct tcctatccat gagcatggaa tgtttttcca tttgtttgtg tcgtttctta 12660 tttccttggg cagtggtttg tagttctcct tgaacaggtc cttcacgtct cttttaagtt 12720 gtactcatca tcactgatca ttagagaaat gaaaatcaaa accacaatga gatgtcatct 12780 catgccagtc aaatggtgat tattataaaa agtcaaaaaa gaatagatgt gggtaaggct 12840 gtggagaaat aggaatgctt ttacactgtt ggtgggagtg taaattagtt caaccattgt 12900 ggaagacagt atggcgattc ctcaaggatc tagaaccaga aataccattt gacccagcag 12960 tcccattact gggtgtatac ccaaaggatt ataaatcatt ctgctataaa gacacatgca 13020 cacgtatgtt tattatagca ctatttacaa tagcaaagac ttgaaaccaa cccaaaaagc 13080 catcaatggt agactggata aagaaaatgt ggcacatata taccatggaa tactatnnnn 13140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnntaaaag atacatcctt 14520 tattcatgcg taagatgaaa tcgagaggtg aaattggata tactgttgct tttaaaaaat 14580 tttaacatat atgtaatttt ttgtacttat ctcattttag cctatataag ttatatatat 14640 tttgtttgtt tgtttgtttg ttttgtttga gatggagtct tgctctgtca cccaggctag 14700 agtgcagtgg tgcaatctcg gctcactgca accttcgcct cctgcattca agcgattctc 14760 ctgcctcagc ctcctgaata gctgggatta caggcacctg ccaccgcgcc cagctaattt 14820 tttttatttt tagtagagac agggtttcac catcttggcc aggctggtct tgaactcctg 14880 accttgtgat ccatgtgcct tagcctccca aagtgctggg attacaagcg ggagccaccg 14940 cgcccggctg taagttatat cttacacaaa tctaggtttc attcagagaa ttatatgcaa 15000 agaaacagtg caataggatt attttaaagc tattgttatt gttagaaaac ataatacctt 15060 taaaattcct tttcacatta gaaatatagt ggcttctccc cagtttagga tagaaatttt 15120 ccttttcttc tccttcttta tactattcag atttgcatgt ttgacagaac aaattataag 15180 agaaaatatt tgaaatgtca catactaaag taaatgtttg aatgtttgaa aattttctgg 15240 ttttcagaga ttttgaattg ctgaatcgtt gtgtaaatta agatgttgag tagtttccac 15300 agagtaatta tttgaaagtc actgaaagca agacacatgc ctaatgtaaa tgtttattgc 15360 actactgtac ctttttctac ctcataaaaa tgagaatagc agtctgtact tttccacttc 15420 gtcattcgta agtctttgca gaaattcata ttttgtttgc ttattatctt cacgctgtaa 15480 atagcttgaa aattctttaa gtggggctag cgatgtatta tggatacatg ttaagtggta 15540 tagaaatttc actttttttt ttttgcataa agagtaacaa gaccagtagt ccatatttct 15600 tcagctctac ccagagaagg gcaatgtagg agggaaaatg aagtttgcaa aatatttcat 15660 agtaggcttt ttcttaaagt aacttcagac ttacagaagt ttaaaaatag tacaaagaat 15720 ccccatatac ctgtcacccc aattcctgaa atattaatat tttaccacat ttgttcatta 15780 tgtctgtatt ctccaagtac gatatatgcc attatatgta atatgtagca ttttatatag 15840 acatagggca tgtatgcact atatattttt ttctgagcca cataaagagt aaaacgcaga 15900 catgacgtgc ttttactcct aaatacttca gtgtgtgtat tccctcaaga aagggcattt 15960 tcttctgtat agctaccgta cacttctaca cttttcaaaa tcagaacatt tacattgata 16020 ccatactatg acatgatctg cagaccattt tccaatatgc cagttgtccc actgtgtcct 16080 ttagtacaaa agaaaaaagt tttttttcct ggtctaggag ctaatcctgg agcacatgtt 16140 acatcctgtt gttttaatct agaaccgttc ctcagttctt tatctttcat aaccttgaca 16200 tttttggaga gtacaatcca tatattttgc agaatttccc ttagtttggg tgtgtctggt 16260 ttttccttat aagattcatt ttatgcattt ctggccagag taccacagaa gtactgtata 16320 tcttaccaga aagcctaagt ggcatttgca ttttctaaat gatcaatttt aatattatat 16380 ggaaagcaga gtcagagatt ctcacatatg tcaagatatt ataagtattc ctgttatatt 16440 tattctccaa ttgctttttc tcaagaaaat ttgtggcctt tcagctagct tttcaaagtg 16500 gaagttacta cataacatta ggatgggagg ggtggggaag agctttatta aagctttaag 16560 attgagcttt tgagtatgtg ttgtatgtaa atgaaagtgg gcattgatgc agggattggg 16620 cctttaaacc tttggccaag aatggtatca attattatta ttattatttt ttggagtact 16680 tctgctaaaa cactgaaatc agtgtgccac tctcctttta gaagttttac acctttccaa 16740 ggtacacttt tttttttgga gacgagtttt gctctgtcgc ccaggctgga gtgcattggc 16800 gcaatcacag cccacttcag cctctgtttc ccagactcca gcagtccttc cacttcagcc 16860 tcccgagtag ctgggattac aggtgcacac caccatgccc agctagtttt tgtagagatg 16920 gggttttgcc catgttgccc aggctggtct ccaactcctg cgctcaatct atccgtcctc 16980 ctcagcctgc caaagtactg ggattacagg cgtgggccac cactcccggc ttccaaggca 17040 ggcatttaaa tgtaataaat agggagataa gcaagaaccc tgttggacct ggtagaagca 17100 aacatttatt agtactatta cgttgtttaa aatattagcg ccttctatat tcatgtcctc 17160 ccagaattat caaaaaacct actctatagt ttatttggct tatatctcag gagtaataaa 17220 attagttaat agtattggca tcgtggttct ttgtgtattc ctcccttatc ccaccccaag 17280 ttgatttcac atgatctctt gatctagtct aagaatgttt atagtgatta cgagaagttc 17340 agattctggc tttaacatat ataattgttt tttaatctgt aaaccaaaga gaatgagttt 17400 gtttaaacta gaaagatggc aagagtagtc tgggaatttt gttccattcc ttaaaagtcc 17460 tataataaaa taaacatatc ttgtgtttta tttttacaat ttttttaaac attagtacag 17520 agtgccactt cttatattct atatcaaata atgagctaca ttttcaataa taacctctga 17580 gtaatttttg gcattaaaat gctgcattac aaaataattt gaggatataa tttataatca 17640 cttatgctaa aatcacctat ttgaaattat gtatgaggtt ttcaaagttt atagtgcttt 17700 ggaaaaaatt taaatgtttc tttgtttatg tatctttatt ataagctgta gcatatatca 17760 tgtagttgtc aaggatgctg atagatactt aatatttaaa ggagacttgt ctaaagttag 17820 ctgtccagga ctagaatctg ggccttttgg taacagctca ttgctctatt tacttaaatg 17880 atgattggat tcgttagaat ttctctattt tcatagctgt ctctatggtt ctatgaaaat 17940 actgtgtgtg tgcttataca tatatgtata cctgtaagta caaagtagaa aatgaaagtt 18000 cattttctgc ttttgacaat tgtaatcccc agagataacc gttattaata tgttgtctca 18060 tgtttggtca tactgttttc tctgtattct gtgtattact gtataaattt tacacagtaa 18120 tttgcatatt aaaaatgctg gtctacacct ggcccttttt taaaaactgc aatttattat 18180 ggccaatttt ttataccagt atatattgat caaccttatt ctttttaact gctgcatttc 18240 attcattacc aatagatgag acatttccat tggtttgaat ttttcagtat tacagataat 18300 ggttcaatta aatatttaag cttttgtgca cttgtagaat taattcctag acatagaacc 18360 cttatatttt gataggtatt tccaaatttc ttcccaaaat gtttgtatct ctttacttcc 18420 actctcaggt ctaataattt tcacttggat tatcatattt cttacccagc ctgtttttta 18480 cactctaaac tctttttctt ttcttttttt ttttgagaca gcatcttgct cttggcccgg 18540 ttgaaatgca gtggcacgac gaccaacctg ggctcaagca attctctcaa cttagcctac 18600 tgagtagctg ggactacaga cacatatcac catgcccagc attttttttt tttttttttt 18660 ggatttttag tagagatgag gttttgccat gttgcccaag ctggtctcaa attcctgagc 18720 tcaagcaatc cacccatctc agcctcccaa aatgctggga ttacaagcgt gagccactgc 18780 acctggccca aaagctcttt ttctaatagc aatataaatt gtcttttaca gactatactc 18840 atatatgttt cttctttcag aaataggtgt taagtgtatc taacatggaa tgtatagcta 18900 taattctcat tgtgaaacca tagcctaatt tatttcatat tacaatttaa aattcatatt 18960 ttttaggaag ttttcttaga ttaatccgcc tagttccagg tgctacagtc ccaagatttc 19020 tttcttttta acaaattaaa tataggtaac atgactagaa ttgtagtcaa agaatattgg 19080 aaccttggaa cttcagtatt tgaactttat tttgaaatat aatttgttat attataaaaa 19140 tattataata tattgcacct ggaagttagg ggcagttttt tttaattctc tttgtatctg 19200 ctacactgta aagtgctatt tatgtaaaaa attcttaata gaagtcttca gttgtaaagt 19260 ctgctgtaca gactttagat cagggattgg caaactatga gccatgtgcc aaatcctgcc 19320 cttcacctgt tttgtaaata aagttttatc agaacacatt cagactcatt catgaacata 19380 ttgtctatga tttattttct gctactatgg cagaattgag ttgttgcaac tgtgtggcat 19440 ccaaagccta aaatatttac tctcctggct ctttgccaac ccgttttaga ttatgagcac 19500 tttggcatta ttatgttttt gttttctttc tatagcacac agtaagatgt tctgcccaca 19560 ttgtgcataa tttatgggtt tattcaagga tttatgcaag tgtagctgca agaaaaaaac 19620 ctagaagtga acttgctagg ttgaagagca tctgtgtatg ttaaattttg ttagctttcg 19680 ccttcccaaa gggattattc catttcatac ttaaactact aattttgtga taggacttct 19740 ttctccatag ctttgctaaa ttaatgcatt cacacacttc atctttacta atctgataga 19800 gggaaatgat attgtggatt tgatttgcat ttctttttat gtgttagctt gagcttattt 19860 tcatatttaa aagccaattg tatttctttt tcttgagcta tcttttaatg tccttcctga 19920 tacatttctg aagtctgtga tactcatata agatatatgg tgaacatgtg tcaaagattt 19980 atttgactct aatgagggaa cccgcctgat gacaaggctg attgagaaga ggatgtgtga 20040 gatgaagtgt atatcatcag tgaaagaaag caaattctta cagggcaaaa acaaaaccac 20100 aactctaagg gttattgttt ctactggaca gaattcattt gcattttacc agataaaaat 20160 tactattttc aatttatctt ttacaaatca ttttctaatt ttacagagtc tattccctaa 20220 tcagtagtaa atagtcttca aaattctccg cagcgtcagg tgactattat gcaggctaat 20280 tgttgacact cgggcttgac tttaagagaa catgccataa tcttttggcc ttacttccaa 20340 gttttggata atttttctta acacattttt ctctaattgc aatgatttca agtgatatta 20400 tttctttttt ttaaattttt ttactattta ttgatcactc ttgggtgttt ctcggagagg 20460 gggatttggc agggtcatag gacaatagtg gagggaaggt cagcagataa acatgtgaac 20520 aaaggtctct ggttttccta ggcagaggac cctgcggcct tccacagtgt ttgtgtccct 20580 gggtacttga gattagggag tggtgatgac tcttaatgag catgctgcct tcaagcatct 20640 gtttaacaaa gcacatcttg caccgccctt aatcccttta accctgagtt gacatagcac 20700 atgtttcaga gagcaggggg ttgggggtaa ggttatggat taacagcatc ccaaggcaga 20760 agaatttttc ttagtacaga acaaaatgga gtctcctgtg tctacttctt tctacacaga 20820 cacagtaaca atctgatctc tcttttcccc atatttcccc ttttctattt gacaaaactg 20880 ccatcctcac catggcccgt tctcaatgag ctgttgggta cacctcccag acagggtggc 20940 ggccaggcag aggggctcct cacttcccac actgggcggc cgggcggagg cgccccccac 21000 ctcccagacg gggcggctgc cgggcggggg cgccccccac ctcccagact gggtggccgg 21060 gcggagacgc tcctcacttc ccagatgggg cggctgccgg gcggaggggc tcctcacttc 21120 tcagatgggg tcgcggctgg gcagaggtgc tcctcacctc ccagacaggg tggcggctgg 21180 gcagagacgc tcctcacctc ccagacgggg cagccgggca gaggcgctcc tcacatccca 21240 gagggggcgg ccgggcagag gcgctcccca cgtcccagac gatgggcggc cgggcagaga 21300 cgctcctcac ttcctagacg ggatggcggc ggggaagagg cgctcctcac ttcctagatg 21360 ggatggcggc cgggaagagg tgctcctcac ttcctagact gggcggccgg gcagaggggc 21420 ttctcacatc ccagacgatg ggcagtcagg cagagacgct cctcacttcc tagtacaggg 21480 tggcggccgg gcagaggctg caatctcagc acttcgggag gccaaggcag gtggctggga 21540 ggtgggggtt gtagcgagcc gagatcacgc cactgcactc cagcctgggc aacattgagc 21600 actgagtgag cgagactccg tctgcaatcc cggcacctcg ggaggccgag gcgggcagat 21660 cactcgaggt caggagctgg agaccagccc ggccaacatg gcgaaacccc gtctccacca 21720 aaaaacacaa aaaccagtca ggcgtggcgg cgcgtgcctg caatcccagg cactcggcag 21780 gctgaggcag gagaatcagg caggaaggtt gcagtgagcc gagatcgcgg cagtacagtc 21840 cagcctcggc aacagaggga gaccgtggaa agtgggagac ggagacgagg gagaggggga 21900 gaccgtggaa agcgggaggt ggagacgagg gagagggaga gggattattt ctgtatgact 21960 taataatgaa tttctaagag gtcacttagc tcactgttgt ctcttctaaa acatactcat 22020 ctttcctttt ctcttctgta ggaactcatt atacaatgac aaatggaggc agcattaaca 22080 gttctacaca tttactggat cttttggatg aaccaattcc aggtgttggt acatatgatg 22140 atttccatac tattgattgg gtgcgagaaa aatgtaaaga cagagaaagg catagacggg 22200 taagtgtttt tagtaaaaat ttttaaaaac atagtgcata attagatctt ttaataatat 22260 atttctgcca atgatctcag gctgccaaat gtttacattt aatataagta aatgtctaca 22320 tttcatatgt ggtacatgtt tttttctttt tctatgttta atttttttag tttacttata 22380 ccctgtaact ttccagaaag gatttcaggt agctaaaaaa caaagaaata caataagaag 22440 acaaaataag aaggaaaggg aaaaatacag cacaggagtt ggggggaaga acaagccaag 22500 ttccagatat ggaggtcagc atgattttgg gctttgagca gcccaccagc taaggcaaaa 22560 aaggaaactc attgcatagc tcttacctat ggaaaaagaa gaaatctact gggggcagat 22620 ggtcttgtgg gattttgctg ttttctttta tctcctttcc cagcatttga ttctgagata 22680 tttctcaatt tggctcccaa ataaagctta ttgagtgttg taatggttta ctgttttttt 22740 taaaaatggc tttaacatat aaaagtacaa cttatggatc ctttttgttt gtggtcgtga 22800 cttactgata atataatcca aaatacattt tttattttgt atttatttat ttatttttga 22860 gacggagtct cagtcttctg cccatgctgg agtatagtgg tgtgatattg gctcactgca 22920 ccctccgcct cctggattca agcgatgctc ctgcctcagc ctcctgagta gctgagacta 22980 caaacgtacg ccaccatgcc tggctagttt ttatacaaaa tacgtttttt aaaaaacaat 23040 tttttttttg gaggtcgggg gactgtcgcc cattctgttg cccaaactgg agtgcagtgg 23100 tgcaatcttg gctcactgca acctctgcct cccaggttca agcgattctt gtactcagcc 23160 tcctgagtag ctggaattat aggtgtgtgc catcatgcca agctaatttt tgtattttta 23220 gtagagatga agtttcgcca tgttggcgag gctagtctca gactcctggc ctcaagtgat 23280 tggctgacct cagcctccca aagtagaaaa tcttcttgaa aaataaaatt ccaaatctca 23340 aaaggcccta tataattttg gtgttggaaa tttacttgtc aatgaaaatg actatttaca 23400 caaattataa gcttccatat taatatatat gtgtgtgaac ctgaaattca aattttatta 23460 tattgtttat gaaaggtaca gcctctgaga ttcatcagat ggtatttacc tttagggcat 23520 atctaaaaat aaaatacagt acatgaaatc cagtgcttta atccagtgat tcttaaactt 23580 tttgctctca gatccccttt aaactcttaa aagatattga agagctccaa ggaggctttg 23640 tttacgtggt ttttatcaat ggatatttac catattagac actgaaactg aggattttaa 23700 aaaaaaataa ttcatttaaa aataacagta acaaaaccca ttacatgttg acataaataa 23760 catttttacg aaactatatt ttcaaaaatt agtgagagaa tgacattgtg ctacatttgt 23820 tataaatctc attattgtct ggcttaataa aacactgctg gattctcata tctgcttttg 23880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24840 nnnnnnnnnn nnnnnnnnnn nnnnnnnaaa tattgattca ctgatttatg tggatctttt 24900 aaatgttgac acttatataa tataatacaa tattttaaaa atcacatttg ttaattttac 24960 ctttgatcta ttcagaaaag actctaagta ttgggaacct atcatcctca cagtgataga 25020 tacaagtttc ctaaaattct gatttttact ggagagctca aattctatca ttggaaacaa 25080 atacacattt atttaactta aaaatgacag gattacttgg tttcattatt gagaaaatac 25140 ctgtcaaatt cccaagtctg gaaaaccatg gtttgatgtc actctttcaa gtaaaaatgg 25200 cattccatgt aagaagtgtc tagtttatta tgcaactcaa ataaattacg caagtgcttt 25260 tctttaggac ataacttcat acatacttcc acaagcagca gatgtgtgta gttatgcata 25320 gttccttatg catggttctt atttcatcac acaaaatatt aaaaagactc agtgattgag 25380 acgtagcagt ttttactgct tcatcaaaga tgctcttatt tgaaactggc ataatatgat 25440 ttatttattt gattttactg ggaagcatgg cagtcaagaa tgtaatgact gccagtacat 25500 ttgagtgcca ctgcttgatt tttgctatgg agtcagcaat tttgccactg gttttgcatt 25560 ttcagtaaaa atgtcaacac agtgaaaaag gcacataatg tcttgtatta ttttgtaaac 25620 agttttatct tgcagacccc ttgaaaaggt ctcggggatc ctccaaggtg ccagtagacc 25680 gtactttgaa aatcactatt ttaatccaaa gtgcctagat cagacacact ataaatcctg 25740 tgtcttgtat gatcattagg taaatacatt tgtacttaga agtatacatt cagagacatt 25800 aacagtattc aggttgggat ttaagtatat tttaaagtgt ggtacctaga gagtatccat 25860 gacactatgt tcataaaatt ttagagaaaa ctgagatcaa aggaaaccaa aacaggctgg 25920 tcatagtggc tcatgcctgt aatcccagtg ctttggaagg ttgaggcaga ggatcgctgg 25980 atcccaggag tttgagacca gcctgggcaa atatggagac tatctctaca caacaaaaca 26040 aaaattagct gggtatagtg tcttgcgcct atagtcctag ctactcggaa agctgaggtg 26100 ggaggatccc ttgagcctgg aagttctaag ttacagtgaa ttatgattgc accactgccc 26160 tccaacctgg gtgaaacagc aagaccctgt caccctccaa aacaaacaaa aaacactttt 26220 ttctctgagt atgtaaatgg ttagtgtaca gtccttgaaa acattgcaaa tagtatagca 26280 atatatgaag tagccagtat gtgtcctagc taattttatc aatcatctct tcctagacca 26340 atcaaatatt tttcaatatt ttgatccatg cttatatgaa caagattttt taaagctgga 26400 aaattccaca catttatata cttactattg ttcttaaaat taattttttt tttttttttt 26460 taagcagagt cttgctcttt tgcccaggct gaagttcagt ggggcgatct cgactccctg 26520 caacctctgc cttccaggct caagcagctc tcgtgcttca gcaccccaag taactgggat 26580 tacaggcata cgccaccaca ctggctaatt tttgtagttt aagtagagat gtggtttcgc 26640 catgttggcc aggctggtct caaactcccg gcctcaagtg atccacctgt ctcagcctcc 26700 caaaatgttg ggattacagg tgggagccac tgcgcccggc ctacattaaa ttttaaagcc 26760 tttctatgtc agtgcatata cccaacctaa ttcttttttt ccgtgaactt ttttgttatg 26820 cttgtagcct tcctacccca gattatttcg aagcaaattg tcattctgta atttcaaata 26880 ttactatttc agtattttac aaaatggttg cagtttaatt gttgttcctt ttttatttat 26940 tagcttgcat atttctatag agagtttacc ccacatcaac catttggatt acctgaagta 27000 agggtggtac aggaaaggga gaaatcttga aatactaggt tccttagcat cctcaaagtt 27060 gaccaatgag attttttgct tgtttggttg tttttttctg tgtcttctgg actcatggat 27120 ttaagtatat ttgtggttta atcatcactg ttattattct tattgatgtt catgttattt 27180 tagattagtg ggagcttttt tagtttgcta tctgtgtcct tcgtcatgtc cttagataat 27240 cctaatccta atcctgattc atcgtagaca tttcccgcag caaacctgga atcagccatt 27300 tctcaaggag ctctctgatt ccattgaagg aaaatataat ataggtacaa tctaggcact 27360 aggtgatact tgttacttct gggttggcta ttgtttctag cctcctaagt ttatatgact 27420 gtactaattt gaattcataa ctatgggact aaacttctaa ttcttaaatc tgcatttcct 27480 ttaagtcatg ccaaaaatct gaacatcaca aacatagtca tttcgtttac cccacaatac 27540 acacatacaa cattgtcagt ataacagtac caacaccatc tccaacaata tgcctactga 27600 aaaattttag gtaatctgtc tccagcctcc caggtagctg ggactgcagg tgcacaccac 27660 catgcctggc taattttttt tttttttttt ttttttaaga gactgggtcc ttgctatgtt 27720 actcaggctg gtctgaaatt tctggcctct aacagtcctc ctgcctttgc cttccaaagt 27780 gcagagatta cagacctgag ccaccacgtc tggcctatcc tttatttatt ccaccaaagt 27840 tatttataca aattactttg ttgtaaagtc ccttggaata gtttcttctg tggcattatg 27900 ttaccagtta gatgcacctt tgattcattt aactttactt caatttttaa ggtttgcttt 27960 ttagatttag ttttgtttta ttatacatat atgaagtatt tccacggttc caaagttaaa 28020 tgaacaaaac aggcatgttc aaagaagtct agtttctatc tctgtcccat ccaacccatt 28080 gtcttcttcc ccttataagt aataatttac atttttaact tgtggtttat cttctgattt 28140 ttaaaaatat aagcataaat atttatattc ctgtctttta gcatgctttt agccatcttg 28200 cttttttcct gtataatgct aaatatatct cattcttttt aattgctgca gaatttctca 28260 ttacataggt atactgcaat ttatttatct gatgctatgt tgatgaacat ttaaatgatt 28320 tccagatttt aggaacggtg atgattgaac tctctgtaca tatatctttt ttacttggta 28380 cactccatca agcaactact taagtgactg actatgatgc tgtgcaagca gttatataaa 28440 gaaaacagca gtgactcagc ctgaaaacgg cttaatatta tcatgttttc ttacacatta 28500 tttttattga ggaaaagcaa catggagttt agtgattatt tttgaaagaa ataacctatt 28560 tctaattcta aagaatggtt annnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nngagtctag ctctgtcacc 29160 caggctggag tgcagtggca cgatctctgc tcactgccac ctccgcctcc cggtttcaag 29220 tgattctcct gcctcagctt cccaagtagc tgggattaca ggcgttcgcc accacaccca 29280 gctaatttct gtatttttag tagagaaggg gtttcactgt gttggccaga ctggtcttga 29340 acttctgacc tcgtgatcca cctgcttcgg actcccaaag tgctgggatt acaagcgtga 29400 gccaccacac ctggccaaaa atatgggttt ctaaagcaac agtcctagta caacagaaga 29460 gaggtgttga ctagttaggg atttaggttt agaagtacat tcttagtaag agaggtgaga 29520 cttaccttct tgtgttttag tatagtgaga tctggatcaa atctattact cttattaatc 29580 tcctaacttc ctacactata tccagtagag gacacttttg ccttacacag taaagaaaga 29640 gcctctggac tctaccaatg ggatcggagc tctccaaacc tgcatattaa aaggcctata 29700 agttttgggg ggtccctttg tccacatgat tattctgtaa tacattgtat ttatggacat 29760 ggtattatta tacacagatc ctgtctttta aagaacatta taatccactt aactgctagg 29820 accagagaat gaccgataat tcaaaccata ttgtcttaca gaagacatat ataaaagatg 29880 gttatgtgta ccaattgagg ttcaaatttg attcaattta aaacaatcta ggccagattt 29940 tatatagttt gtggaccctt tgcactcaaa tctcaaggtt cttattaaaa tgcagatctt 30000 ggctgggcac ggtggctcac acctgtaatc ccagcacttt gggagcccaa ggcaggtaga 30060 tcatttgagc tcagaagttc aagaccagtc tggccaacat agcgaggccc agtctcattg 30120 aaagaaaaaa aattttttaa taaaaaataa aagcagatct tgggtaaaga catgtagtct 30180 ggtttacagg tattaacaac tgtctgtaat gtagtgattt tgctccagac ttaccttttc 30240 cattatttag ttctgaaatt actgttctat gtatggtaaa tgagaaaaat tgctagattc 30300 tagaactgtg gcttctattc atagttggaa aaatgaagca taaacatttc taatttcaga 30360 tcaacagcaa aaagaaagaa tcagcatggg aaatgacaaa aagtttgtat gatgcgtggt 30420 caggatggct agtagtaaca ctaacaggat tggcatcagg taaagaaaat ttttcaagca 30480 atcctttttt agttaacaga agtataaact gttcttccct ccttccctca attttttttc 30540 aggtaccatt ggattttaaa aagcatttgt ttctcttctt caaaaaatct ccttaaatat 30600 aagactagga ggcagaggct tccaagtcta gtcttggctc tatcacttta cgtgtttatc 30660 cagcttggtt gatctttctg gactcagttt ctatatctgt aaaataagtg gtttggatca 30720 gatgatcaat aaagtatctt ttgatattaa catcgtaata aatagctaat atttcttgag 30780 tgcttcctat gtnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30840 nntggaagat tatgttcaga agaccataaa aattaaaatt tttgtggaga ataaagtact 30900 gataattcta attggcatgc atagtaattt tatggcctct gtgtatgtaa cccactgatc 30960 tctttatgta agaaggaccc agatttgacc ataaatttgt gtatttttta tattctcaca 31020 ataaaataat cttgatatat ggttttctgt aatttaagaa aatattattc ctatgagttt 31080 caataattat ttctaatgga cattaaattt taatgaaatt gacatcattt ataagtctgt 31140 taattaagtt atcgattgaa aattagattt gtgaacctcc tgccaagtag ctgtcttttg 31200 aagatatttt agtatctttt aaacattgtt tttcagatca caattaattt gaatgatgta 31260 actttttaaa attccaaaca aaaatagcac ttttattgta aaaaataact ctttacagtt 31320 tataactaaa atttgaaaat cttaaattta tatgtagttc ataaatgacc ctttatttag 31380 gagtctcctg ctttctactt gccttttaac tagattgttc tcgactccca aaaaattgac 31440 ttaatttttt taccatctcc aacatgtttt tataggggca ctggccggat taatagacat 31500 tgctgccgat tggatgactg acctaaagga gggcatttgc cttagtgcgt tgtggtacaa 31560 ccacgaacag tgctgttggg gatctaatga aacaacattt gaagagaggg ataaatgtcc 31620 acagtggaaa acatgggcag aattaatcat aggtcaagca gaggtaagtc ttgctttgtc 31680 tcaagatgaa ttaataattg atatagcaaa atgtttccaa ttcatttaat tatagaacta 31740 atcacatatt agatgattac atacacatca aatggatcca ccctcaacac attgcagcaa 31800 gaaagaatta agtgcaatat tgtttcaagt agctttttta ttagttaact gcatagtcat 31860 ataacaaatc ctctggattg tggtgcaaat atatttgagc tgtagtagaa aagaagtgat 31920 agttattgca gtaagatctg tgtaaagtta ctaagaagtc aagttattaa aactaatata 31980 ttactaagat tgggaagttt gaattatgaa agtattatca aataatttag taaaatcaac 32040 ctacgtagag atacattgaa gataatcaga catttttatt tgtggcatta cagcatttaa 32100 atgattgatt tactatgatc tacaaagaac attttagaac ttaggatgtt acatgtatat 32160 tttttacatg atgacatgga tatatttttt aaattttgtt ttagctgaac tttagagcta 32220 aaaggtatac atttgcggta agatgagtag tatgctgttt ctcacctggc ttaattgaat 32280 tgagtttaat gatctggaaa gttgcagcag aatgaaatct gagtggtgat gcaatttgtt 32340 tccactgttt ccaaaaagtg gtttgtaggc agagattgaa gtatagctga gatgtgttgg 32400 taacaagact ttagggatta ggaaaaagat taaatgtgct cagggttcct tggtatatgt 32460 aggcattaat ttttggactc tacttaaata ttttgttcat ataaagtttt tattattgtg 32520 gaaataaacc aggagacttt tacacatttt actgaagttt cttttctttc tttttttttt 32580 tttttttttt tggccggtgg gatggagtct cactctgttg cccaggctgg agcgcagtgg 32640 cacgatctcg gctccctgca acctccgctt ctggggttta agcgattctt ctacctcagc 32700 ctcccgagta gctggtatta caggcgtgcg ccaccatgcc cagctaattt ttgtattttt 32760 aatagcaacg gggtttcacc acattggcca agctagtctc gaactcctga cctcaggtga 32820 tccacccgcc tcaacctccc cagtgctggg attacaggcg tgagccacca tgcctggccg 32880 tttactgaag tttcttatga caagcatttg cattagaggt gcaatgtaaa ttaaattcat 32940 actctcgaac tattttcttt ttagggtcct ggttcttata tcatgaacta cataatgtac 33000 atcttctggg ccttgagttt tgcctttctt gcagtttccc tggtaaaggt atttgctcca 33060 tatgcctgtg gctctggaat tccagaggta agccaagtaa tatttagtgt cattaaacat 33120 tattatgatg cttatctttt tgaccttagt gataataaaa gttggctttt ctggagggag 33180 gggatagttt gttcataata tgaaaaaaaa atttttttaa gtataagctg atggtagaca 33240 tcattgaaaa atattgttcc ccatagtcat ttggtcattt actgtgaagg ctgatttttt 33300 ttttctctca ccactaattt aacacatgac taggcaaatt ttcagactat ttagttaaac 33360 atcaagagcc tggaagaagt atcttgtgac ctaatgttct ttgacgggtt agttgttact 33420 ttgctgttat gaccctgaat tttttttttt tgagactgag tcttgtgctg tcgcccagac 33480 tggagtgcag tggcgcaatc tcagctcact gcaacctctg cgtcccaggc tcaagcaatt 33540 cttgtgtctc agcctcctga ggagttgcga ttgcaggcac ctgtcaccat gccctgctaa 33600 tttttgcatt tttttgtttg tttttttttt ttagtagaga tggggtttca ccatgttggc 33660 caggctggtc tcaaactcct aacctcaagt gatcacccgc ctcagcctcc caaagtgctg 33720 ggattacagg tgtgagccac cacacgtggc tatgaccctg attttgattc attcactttt 33780 tataattacc ttttgattag ataagttaat tattcttgaa tttggccatt ttatgctttg 33840 agaaagtagt taatcacagt gggtcaacag tacaaacttt tgggttttat ttttcatcac 33900 aataaagtag agttatacat aggattgatt gaacttgatt tgaacttatc tcttctcttt 33960 tatttttctg gagttaaata agttaccaac tttttcctaa tacatttctt tttaaaatgg 34020 aattgtattg atcctttaag tttgtattaa gaatatcttt cataaaaagc aatatcatgc 34080 agtatataac agttgttact cattcttgat acataaaaaa ctattgcaca taattacagg 34140 acctcagaga aaacataata ttcttatttc taacataatg gccaaaatat atttaaaata 34200 ttatgcttat ttttacaaca gaaatattca aatttgccct ttttttgggt atgtaattat 34260 aatccttata attaaggtct gtattcattt taacatggcc tgatattttg attttggcct 34320 gagatagtgt tgccctctct cctttcttgg gtagagaatt agattataat atcaatttat 34380 tatatgtagc ataataggca agttttcgaa aaattaactg taaatttttc tgtagactgc 34440 taaaatttgc aaggttgttt ttgtgcataa aacaagaaaa taacttggat tcgttacatt 34500 ctcatgtttc ttaaaggaca ttaagctgcc ttaatctttg ccttgtagat taaaactatt 34560 ttaagtggat tcatcatcag aggttacttg ggaaaatgga ctttaatgat taaaaccatc 34620 acattagtcc tggctgtggc atcaggtttg agtttaggaa aagaaggtcc cctggtacat 34680 gttgcctgtt gctgcggaaa tatcttttcc tacctctttc caaagtatag cacaaacgaa 34740 gctaaaaaaa gggaggtaag tgtcttttgt agttaatttg actgaaaaat atatattata 34800 tagtatttat ttaagtaaag aatttcttag tgtaaaaata ataaattctg tattcagata 34860 aaaaattttg agatttgtgc ttctgttttt cctgaataat ctataacatc tttctagaat 34920 ccattcccag tgctgctcag ttcgtcttac attttagaga agctttagat agacagctgg 34980 tgtccattgg gtttcagctg catttcacga agatcttcct gttatcactt taccttacat 35040 ctttcctctt ctgaagtgtt ttctaagctt agctttgttt ttcactctta ctttcaacat 35100 taagaggttg ggaaatctta atagctatgt tttcctcctg gaggcagtgt ctggtgccag 35160 tgtaagtggt gtgtgatatg aaaaatgcta tccagtgcta tggggaagtt ctgagggcct 35220 ttagaagctc ttgaagttta aatcagaaat tcacattaaa gagattacag gaaatccttt 35280 tcatttgatt gtttaaggca atttccttta ccatttcttt aggccagcct gagatcttct 35340 acaagacctt gaaaccttat atatattatg gatttcctct gatgtttcca tattgctctg 35400 ggcattttcc tgaatccttt atattagctc tagactttgg gagcccagtc ccttcctatt 35460 ttccaaatct aaatctacag ccctagatgg tacagagatc tttgagtttt taagatatga 35520 ttttttgaaa aacatctcat taaatactgg cagaaccttt tcatcttgtt gagtttttta 35580 atgtactgta accaaaaaag tagaatattt tatcaaactg tttaatcttc aattgaaata 35640 attctagtac attttaatgt tcgcattaaa atattgtcct tgcattggac gtagatatcc 35700 caaaagtgga atacttcaga ttgtcgtagt ttcatctctg aataattgtg attccagtac 35760 tttataacaa aaatagctag cattattgat tactttctgt gtatctggta ctgtggcaga 35820 tactttactt ggattttaat acttaatttc acagtaattt agtaatatgg ccctgttatc 35880 ctcatttagt gattagtaaa ctagggctga aaacagctaa ctaacttgcc cgagactaca 35940 tacctagtaa gtggtggaac gtaggttaaa attcattttt ctttgacttc aaagtctgtg 36000 gtcttaccta cttacattac tgcccttacg actatgtggg tatatatttg tgtgtgttca 36060 aaacaaactc aaaaccatcc tgtagcgtag caagttagtg gctaagatga agctagagca 36120 tttgcctcct caattcaatt ccattacttt ctgttgtacc tttatatttt ttggtaagac 36180 ttttacttat tctaagttca aaaaatgtaa tttattagat gtttgagaaa ttaagtttac 36240 ctaaatttta atgttcatac tgtagtgatt agttaatgtt taatacgttg ttattctgtc 36300 accttagtgt atatataaat ggcaagaatt cacggttagt tgaaagcatt aaggtcccat 36360 agttttgtgt agacaagagg ggagagcgtt gatattttta aattaaatgc ttcttagata 36420 cgtatgaaat ggattaaaac atgtatatga gttatagata cctaggtgtt agtttggttg 36480 taaattcagg atcaggacat tcaaataaat atgtttgctt tcctcttagt ggaggaaaaa 36540 aaaaagaagc taaatttgct ccctttcctc cccaaataag cagagtctac attttaatgc 36600 caacaatttg attaaaacaa atatttattt atttttaatt caccaaactt ttataaagta 36660 tttactggtg ccaggcactg ttctaaagca ctctgtatat atttactcag tccttaagag 36720 ctaagtaata ttatcacgtt tccattttag agaaaactga ggcacatata ggttaggtta 36780 tctacccata gccatacagc tagtaagtag cagagccatg atttcaacac agcagcctga 36840 ctatggagtt catgatctta accatttaca gcttaatttt tattatttat aatttctctt 36900 ctggaaatgt aacaattgac catttgaaga aatactttag gtagctttgg atatttgctg 36960 tattaaagta gtgaaagtaa tacagacact tggctgggcg cggtggctca cgcctataat 37020 cccagcattt tggtaggttg aggcaggcag atcacctaag gtcaggaatt cgagaccagt 37080 gttgccaaca tggtgaaacc ccgtctctac taaaaataca aaaattagcc gggcgtggtg 37140 gcaggcgcct gtaatcccca gctactcggg aggctgaggc aggagaatca cttgaaccca 37200 ggaggtggag gttgcagtga gctgagacga cgccattgca ctccagcctg agaaacaaga 37260 gagaaactct gtctcaaaaa aaataaagga atacagactc ttagaaaaat aattacaaat 37320 aaaaccctag tgaaattata ggtatagtta ggtatagttg gcttacaggt gggaagtaga 37380 ccattaccaa ctgatagact ggggagctgg agagaggaca cggaagagtg tccttggatt 37440 tttcnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 37500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 37560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 37620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 37680 naaaattgtc tatattcatt gcctcctcct ctttacaccc tattcacatt agtatatctg 37740 gcaaaaattt tttttaactg aatggtaaat gcatgactga cctttcaatt aaagccagga 37800 gaaagaaaca aatcttaata gaagaaatga atagttaccc tttgcttagg gagcaaggaa 37860 acatgcaagt taaattcaga aaatccattt ggaaaattca agtaacatga agaattttta 37920 tttggtatgt ttgaatttct atgaaattat gaaataagcc atatcctctt tctaggtgct 37980 atcagctgcc tcagctgcag gggtttctgt agcttttggt gcaccaattg gaggagttct 38040 ttttagcctg gaagaggtag gtgaaaagaa tacaacaatt aaaattatat ataattacca 38100 ttacaaatat atttcacaca tttcagtttt gtaggtgatg taataggtag agactttgtt 38160 ttcaaattta tttttctaaa gttgttttcc actcattctt aataaaaagt aaatgttatt 38220 catgctccat acctggagga aactttttaa aaatttatta atgtatgaat gttagtaatt 38280 atttaaaatc taactttgtt gacatattta aaagtaagaa gatgtgaatt tgacttaata 38340 gaggacatgt gaaacaatct atttccattg gctaaattct gtatttttag tagagatgga 38400 atttcaccat gttggccagg ctgttttttt gtggggtttt tttgttttgt tttgttttgt 38460 ttttgttttt gagacggagt ttcactcttg ttgcccaggc tggagtgcaa tggcgcgatc 38520 ttggctcact gcaacctccg cctccagggt tcaagtgatt ctcctgcctc agcctcccaa 38580 gtagtttttg tttaaaaaat tttaatcaat tcctatgttg agttttaaag tttttcccat 38640 gtgattattt ctgatacagt tagtgatgtt aaagaaaata attttagtga cttcagtgga 38700 ttattttgtt tttgttttct taataggtgt ttaagacttt tctttttaca taaaaatgta 38760 accaggaatt tttttttaat tttttttgac aaataataat tgtttttgtt tatggggtat 38820 aatgtgatgt gtctatacat gtatacattg cggaataatc aaatcagagt gattagcaaa 38880 tccctcaaat atttattatg tccttgtggt ggtgagaaca tttaaaatcc tcttttagct 38940 attttgaaat atataataca tattattaac tgtggtcatc ttactgtgca atagaacacc 39000 agaacttatt cctcctctgt aagttcatac ccgttgacta atgtctcccc tttccctgtt 39060 cacctcccca acccctagcc tctggtaacc cctattctac tctctacttc tatgaattta 39120 actcttttag ttcaagatgt ttttaaatgt acttttttct tttagttgtt tgtattcttt 39180 tttttttttt aatgtagaag aggcaaatta aatgcattat aagttaacag gagttggtga 39240 tggtacattt atttttaact accatgattg aattgaatgt gaaactcatt ttgaatataa 39300 aacagcacta ggtattctat tagtatttat tagacattta tgatcaattg atactgtcaa 39360 tttgtaatga tgatcaccat ctccaaaaat aataataaca tcaatttttc ttattacagt 39420 aaaatccatt acatgtaaat tctaactaca gcaaaattta gagctaggat atttaccatt 39480 caagttataa tatatcagaa acatcttata aaattatagc attaattttt cttttccttt 39540 tctttttttt aggttagcta ttattttcct ctcaaaactt tatggagatc attttttgct 39600 gctttagtgg ctgcatttgt tttgaggtcc atcaatccat ttggtaacag ccgtctggtc 39660 cttttttatg tggagtatca tacaccatgg tacctttttg aactgtttcc ttttattctt 39720 ctaggggtat ttggagggct ttggggagcc tttttcatta gggcaaatat tgcctggtgt 39780 cgtcgacgca agtccacgaa atttggaaag tatcccgttc tggaagtcat tattgttgca 39840 gccattactg ctgtgatagc cttccctaat ccatacacta ggctaaacac cagtgaactg 39900 atcaaagagc tttttacaga ctgtggtccc ctggaatcct cttctctttg tgactacaga 39960 aatgacatga atgccagtaa aattgtcgat gacattcctg atcgtccagc aggcattgga 40020 gtatattcag ctatatggca gttatgcctg gcactcatat ttaaaatcat aatgacagta 40080 ttcacttttg gcatcaaggt aagtgctaat gtgaggtgat atttgggtaa ttttggcatg 40140 ttcaaaactt atatgtggaa tgagagaggt tgttgtttca taaatgactg aaaaaagtac 40200 ttatcttttg agtttaattt taagtaatga aaaagataat tccttagcat atattgttga 40260 ccatgttatc tgttgctatt taacaaatta ccccccaaaa cttagcagct taaggtaact 40320 acttattttg ttcttgatat tgagtcaacg acttgggaag ggctcaactg ggcaattttt 40380 gcttgtggtc tttcatatag ttgttattag acatggcgag ggctaatcat ctcaaagctt 40440 ctttttttcg tttccttttt aaaaaactgt ttttgtggat acacagtagc tatatatagt 40500 tttggggtat atgaagtatt ttgatagagg catggagtgc ataataatct cagggtaaat 40560 ggagtatcca tcacctcaag catttatccc ttgtgttaca aacaatccaa ttacactctt 40620 aattattttt aagtgtacaa ttaaattatt gaatatagtt caaagacttc ttcattcatg 40680 actagcacct aggctaaaaa aattcagaca cctgggctcc tgggatcaat cacgcatact 40740 gtgtctcttg tgctcactcc cgctgtctct ctctctttct ctcgcttcct ttttcctctc 40800 tctctgtggt tttctagggt ggtggcctca gggaattgga tttcttatat tatagctcag 40860 gattcccaag agggctgttt ttaatgtagc caaagaagtc ttgcagcgtg acttgtttta 40920 ttctattcat tgaggtagtc acagaggccc gaccacattc agaggaggga catacacttg 40980 ctgggacaag tgtaagagaa ttcatgatca tgttttaaaa ccacttttat tagtttccta 41040 ttgctgctgt aataaattac cacaacttaa tggcttaaaa gccacacaaa tttaatatct 41100 tacagttctg caaatcaaaa gtctgaaacg gatctcactg tgctaaaatt aaggtgttcg 41160 tagggcattc tggaggctgt aggagagagt cttgtttttt gccttttctg gctattaaaa 41220 gctgccagca ttccttggct cctggctgtc tatttgcatc ttcaaagcca gcagtagctg 41280 gtcaagtctt tctcttgtct catcaccctg acccaaactc tgctaaatct cccttccaca 41340 tttgaaaaac ctttgtgatt actttaggcc cacgcagata aatcagaaaa taatctcctt 41400 tttcaaggtc agttgcttcg aaactttctt tctgccacct tgattcctcc ttgccatgca 41460 acgtaatgta atcacaggtt ctgggaatta agttatggac atctttgatg agccattatt 41520 ctgcctcata ccagtatagg gtattagctt gaaaggacac tgcagactca gttaaattac 41580 tagatctata aatacatgcc tttttccatc aagaaattaa ggcagctggg tcttatgccc 41640 tgggacattg cttcttttgg atttataaaa taacaaaatt tgttgattaa tggtctatca 41700 gtaaatataa tttcttatgt gactatcagt gatatatatg gggaagcaca tatcagctta 41760 ttcttgttct ttaaattact acccctgtac ttcatgtaat agtatttgct agtgatgatg 41820 tgcttttaca gatgtaaatt aatgtggaat aacagctttg tttctacaaa attagagtgg 41880 ttttagtttt tgaaataagg tctcttttct cttgtcctaa gtctgtagtc cactgagtat 41940 ctagagttaa ataatagaaa agcctggcca ggcgcagtgg ctcacacctg taatcccagc 42000 tctttgggag gccgaggcgg gcagatcaca atgtcaggag atcgagacca tcctggctaa 42060 tgcggtgaaa ccccgtcttt actaaaaata caaaaattag ccaggcgtgg tggcaggtgt 42120 ctgtaatccc ggctactcga gaggctgagg caagagaatc acttaaaccc aggaggtgga 42180 ggttgcaatg agccaagatc acacccactg cactccagcc caggcaacag ggcaagacac 42240 tgtctcaaaa aataataata agaagaaaat aataatagta atagaaaagc ctaaacattt 42300 tacctttttt tcttagggaa tcaagttaaa agagctgtta aagctctttt tcctacaata 42360 agtaagtgtt gggtaaatcc caactttctc acagtcagtt gaactacaag aagctggagg 42420 caattggcag gcctttgtta agtcccacct ttgactcagc tctggctgaa ggatcatacc 42480 tggcaagaga gtgtaaaaca cactttgatt ttttctattg tttatccttt taatgatcct 42540 aagagactca agagtacatg ccatcatttt gtgtttggct catttcatat tcagaggagt 42600 ttattactct ttcagtagtt tgtttgttcg tttgtttgtt ttttgagaca ggatctcgcc 42660 tttttgccca gactagaggg cagtgttgca gtcttggctc actgtaacct ccacctccca 42720 ggttcaagcg attctcctgc ctcagcctcc caagtagctg ggattacagg tgtgggccat 42780 cacacccggc taatttttgt gtttttagta gagatgtgat tttgccatgt tggccaggct 42840 ggtctggaac tcctgacctc aggtgatcct ttgggaggcc ttggcctccc agagtgctag 42900 gattataggt gtgagccact gaacctggcc tctttcagta gtctttaaat gatcttgctt 42960 atggtgcttc ttatccctgt ttattatcct tattaaattt aatcaataaa tatttttctc 43020 tttttaattg attcatataa atagacttac ctgagagata taggttcagt tcagagcacc 43080 acaataaagt gaatatcata ataaagcaag tcacataaaa gtcttagttt cttagtgcat 43140 ataaaagttc tgtttacact atgctgtagt cttatgtgta caatagcatt atgtctttta 43200 aaaaagtaat actttaattt aaaaatactt gattgctaaa aaatgctaat agtaatctga 43260 gtcttcagtg aattgtaatc tgttttgctt ctgtagggtc ttgccttgat attggtggtt 43320 gctagaggta ggactggctg tagcaattct taaaataaga taacagtgaa atttgccgca 43380 ttgattgaca ctgcctttca tgaaagattt ctctgtagca tgtgatgctg tttgatacca 43440 ttttacctac agtagacctt cttttcaaaa ttagagtcat cctctcaaac cctgctactg 43500 ctttatcaac taagtttaag gaaaattcaa aatcttttgt ccttttaaca atgttcacaa 43560 catctttacc aggactggat tctacctcaa gaaaccactt tctttgctca tccataagaa 43620 gtaactcctt atacattcaa gttttttaaa tgagattcta gcaattcagt cacatcttta 43680 ggctacgctt atcattctag ttctcttgct atttccacca ctctgtagtt acttcttcaa 43740 ctgaagtctt gaacccctca gagtcattca tgagagttgg aatcaacttc ttccaaactc 43800 ctgttaatat tgatattttg acctcctccc atgaaacgtg aatgttctgg atggcatcta 43860 gaatggtgac tactttttga acattttcaa tttattttgc ccggatcaat cagagaagtt 43920 gttatcagtg gtgggtttcc aagttgtcag gggcgaacca tacagatctt cagcaacctc 43980 aactcttgcc ttctcagagg aaagaattct acggagggac ataaggcaga aaaagagact 44040 gaggcaagtt ttagagcagg agtgaaagtt tattattaaa aagctttaga gtgggaatga 44100 aaagaaatta aaatacactt gaaagagggc caagtgggca tcttggaaga caagtgcccc 44160 atttgacctt ggacttaggg ttttatatgt tggcatactt ctggcatctt gcatccctat 44220 tccattgatt cttcttttgg ggtgagttgc ccacatgctc agtggcctgc tagcacttgg 44280 gaggggagtg tgcacagtgt atttactgga gttgtatgca tgcttacctg aggtgtttgt 44340 tgcttaccag ccaaatgtcc ctaggaggtc atattcataa actccatgat tttgcctcta 44400 aatgtgcatg cttgagccca ctcacccaac tcctgggatc ttatcggaaa gctgccgatc 44460 gctagtttca ggtgtttcta tctattggaa gatggccttt ccctgatgct ggctgcaacc 44520 aattattact ttagagagag agcatgagag ctgtctcacc atcatcacct gatggttgcc 44580 tgacattcct ggtggggttg ggaggatgcc tgtcctgccc tgctcatgcc tgactagcta 44640 cctgctgtaa caaaagtact atctatggta gctgtagcca taggaaatgc atttcttcag 44700 taaaacttaa aagtcaaaat tagtctttaa aacaacatga atctccttgt acatctccat 44760 cagagctctt ggaagaccag gtgcattatt agtgatgagt aatgttttaa aaggaatctt 44820 tttgtctgag cagtaggtct caacagtggg cttaaaatag ttagtaaacc atgctgtaaa 44880 cagatatgct gttatccagg ctttgttatt ccatttatag agcacagaga gagtagattg 44940 gcataattta aggattactt aaaaaaaaag tctttgatta ctctcaaaaa aaagtcacgt 45000 ctctcacttt atatcaacag ctaaaaatgg ccaggtattg tggctcacgc ctgtaatctc 45060 catgctttgg gaggccaagg cagaaggatc acttgaggtc aggagttaga gactaacctg 45120 ggcaacatag taagacccat ctctacaaaa aaaaaaaaaa aaaaaagaaa gccaggtgtg 45180 gtggtgcacg cctgtagtcc cagctactca cgaggctgag tcggcaggat cacgcccagc 45240 caagagacgt gacttctgct ttcagttgta cacttagaga ccattgtagg gttcttagtt 45300 ggactaattt caatatcatt gggtctcagg gaatagggaa gcctgagaag agggagagac 45360 aggggaacag ccagttagtg gagcagtcag accacataca acacttatta agttcacttt 45420 cttctatggg catggttcat ggtgcagtaa aacaactgta acaggaacat caaagatcat 45480 taatcacaga gcactgtaac atataataat agtgaaaatt ttcaaagtat tgagagaatt 45540 agcaaaatat gatacagaga cacaaagtga ccacatgctg ttggaaaagt agtgctgatg 45600 gactagcttg atgcaaggat gtcataaacc tcaatttgtg aaaactgcaa catgtgtgaa 45660 gcacagtaac acaaagcata gtaaaacaag atatgtctgt atatcagtca aaatattggg 45720 caactctgat aagtttgtcc acttaacatt gtaccactta agatgaatag catctaccat 45780 ttccgtcatt tgtaaatata taggaggaca taatcacata atcttgaagt aaaagacagt 45840 gcttaaaact gaatcagtta agttttatga aaaatacttc atattgtact tttaaaaata 45900 tatatttttt aatttcaata gcttttgggt tacaagtggt tttggttacg tggatgaatt 45960 ctataatggt gaagtctaag attttactgc aactgtcacc caagtagtat atattgtatc 46020 cagcatattg tccttttttt tttctttttt ttttttcatt tcaccatgga ctaatgaaaa 46080 ttttgttagg gactgacatt agggcaccct tgagctacct tgagctaaag gaaataaccc 46140 ttgaattttt ttctgtttgg cctagagaat gtggtttgtt ttgtaactga attcatggga 46200 ttgttaaggt acaagatttt gctttagttt tatttgtact aggattttgc tatattaata 46260 caatgtgaaa agaatcaaaa gtgttagaaa taaatgcata gaatgtaagt ttcaggcatg 46320 tgagtagagg atctctgctc cataaagagt tctgttgttg ttataggttc catcaggctt 46380 gttcatcccc agcatggcca ttggagcgat cgcaggaagg attgtgggga ttgcggtgga 46440 gcagcttgcc tactatcacc acgactggtt tatctttaag gagtggtgtg aggtcggggc 46500 tgattgcatt acacctggcc tttatgccat ggttggtgct gctgcatgct taggtaatat 46560 ggctgtgtct gcctgtgtgt ggatgtttgc aagtctgaga gagccaagag aaagtgggac 46620 acattcttgc ttaattggtg ggcggattgg ttgagtaaag gagggtgcca ggaggagatg 46680 ttttaacaga taagaaacag tagtactatt agggtattat acagtaccgg ttttctgtct 46740 tacaacattt gttaatacaa gaatttaatg gcattagcat attgtaatat aacttaatac 46800 actatggcag aagccatcta agtacaacat aagcttaatt tgaatcctga ccaaagatgt 46860 ctttgattct ttcatcgtta aggatcttgg cttacctata acaactatag cataatacct 46920 aagattagca ttgcaacaga gtttcagagt aggtttactt tggttctgaa atgatttatt 46980 gttagcctta gtaaaagatg tatttaccca tgctccatca tctaaggtat atttgtaaca 47040 aaatgagaaa aggtaacttc attttaatga gaagaaaagc aaaataccta cattaagtac 47100 ttgagtctat ttaatgtctg ttagggcagg aaaaaatggt tattgctttt catatttaaa 47160 atatcagcta cactctggtg ataatattaa tggttgccat tttgaccagt tttgtttagt 47220 gaataaaaat tatgtgatta ttgatcttta aaaatgtaat atcaattaaa aggaaaggac 47280 agactcattt tcaccaaagt agcaagtatt tattaaatgt ccactttctt tttagcattg 47340 tgctagatac agtgcataat acaaaaagaa catggaccca atctcgactc taatcaagtt 47400 gaggagacaa gatgaacact gagaatacaa tagtgaggaa tactaacaaa tatatacaag 47460 gttaaaagag tctaagtatg gtaggaatat aggggaagaa agagctgaag tacttcagga 47520 agagtagaac atgaggcttt atttaaaaga ttagcagaat ttaaggaaaa ggtgactttg 47580 ttgaagatta taatgtgaag acaaaggaac gaggatggga ataaattttg tattcatgag 47640 gctttgaaga aattgactct agagagtata ttttgggtac ttttgggaaa tgaagttgga 47700 ttagtgagaa ggaacagatt atgaaaagac aagaaacctg attaatgtca ggatgatttt 47760 atatttgaag ttggtcagat ttatggcagt cctggctttg ccatttttag tttgatgact 47820 ttgagaaagt tccttcttga agttttaatt ttctgtatat aaaaagtaat aacacctggt 47880 gatctgctag gtttgttttg aggattatat gagataaaat gcatgcaaaa ctgttataat 47940 agtgcctggt aaaataagtg cctagtttta aaaacaagtc tttgtaaact gcttaggaca 48000 tgcctggtat agggtaggta tgtaatacat agtaggtagg atctgtctcc ttgctatttt 48060 taggtaaaaa aacaaaagga agagcttcag cttaatacag tatgaactga cgagccctgg 48120 taggtttttg agcaaaagag caacacagta aaagtagtac ttaggaaaga ttaacaaggg 48180 aacatggctt atacagtggt aatggggcct ggagtcaagg aggtaagata aaatggtatt 48240 ataattaagg aatagccagg cacgatggca catgcatgta atgccagcta ctggagaggc 48300 tgaggtggga ggatcatggg agtccaggag tttgagacca gcctgggcaa ctgagtgaga 48360 ccccaaatcc taaaaaatac aaagtaaaaa aggaataaag tcatgagggc ttggactgga 48420 ttgataacag tgagaatacc gagaaaggga ccataggcag tgtgaacgca gctcactgca 48480 gcctcaaacc ccagcccaaa cgagcctccc acctcagcct cccaagtagc tgggaccaca 48540 gacatacacc accatgcatg actacttttt ttagttttta cttttgtaga gacagggtct 48600 cactgtattg cccaggctgg tctcaaactc cttgacttaa gtgatcttcc tgccttggcc 48660 tcccaaagtg attacaggca tgagccacag tgcctggccc aaatagtttt ctgtgagtga 48720 atattacttg catcgttaat gtaaatcaaa ggcatcaaag tattttactc tttttgaaaa 48780 aaatttagag gagaaattta ttatattaat attctaccca tatatgagtt taatttgtaa 48840 attgtagcaa agcatgatgt gctttactaa attcctttat aattagaata agcttttata 48900 agggtgaaat tatgtctttg ctacagcact aaaccaaaat ggcaaaattg ttttagtcgg 48960 taagctttgc ttttttaaaa tatgaaataa acaggttttt aaaatgttat tttaatagtc 49020 ttctctgtta taaacaaaga aaattggtgt ttctctagag cttattaaaa gtagtgatta 49080 ttgtcctaaa agaggagtag cagttttaga tgctaatgct tttccctgac tgagttctat 49140 ttgccattta gttttaactg cctagtgcaa aaattctaat aaaatgtaat gatgaggatc 49200 ctgtccttcc tgaccagtgg gtgcttactt ttttcaggtg gtgtgacaag aatgactgtc 49260 tccctggtgg ttattgtttt tgagcttact ggaggcttgg aatatattgt tccccttatg 49320 gctgcagtca tgaccagtaa atgggttgga gatgcctttg gcagggaagg catttatgaa 49380 gcacacatcc gattaaatgg ataccctttc ttggatgcaa aagaagaatt cactcatacc 49440 accctggctg ctgacgttat gagacctcta aggaatgatc ctcccttagc tgtcctgaca 49500 caggacaata tgacagtgga tgatatagaa aacatgatta atgaaaccag ctacaatgga 49560 tttcctgtca taatgtcaaa agaatctcag agattagtgg gatttgccct cagaagagac 49620 ctgacaattg caataggtac cctttcaaaa atatatatat gtatatatga gatggatttc 49680 tggaagaaag gaaagcaata agcagtaaca tttaatgggt cggatttgtg ggggcaaggg 49740 acattatttc atgtccctta acatcttctg ttctttaaga aaggaaggta tgcttcagtg 49800 gatgattttc tgctatatat cacaaaatct gtatttcagg tttgtctttt gatccggcat 49860 gtaccagaaa ttggagtcag attattttcc cactcagata agcctagata agttgatctt 49920 ggttattcaa aacagcatgt aatataagac cttagctaaa tgcattcagt caaatacatt 49980 cttgtattta ataaagttgg cttattggaa tacaagttat tgaaaatctc atcttcatca 50040 gtctctttca tattagaata acactgtttt gctttatcag tctttggggt tagaattata 50100 atattaattt ataatatctg atttaaagtg acaatcactg agatttttat ttctgatcaa 50160 atgccaggtt gaaaaagtat aacgtatcag tcctgttgtg ttttatgcag actttcctga 50220 aaatactgtt taaaggtatt agccatagtg tatttcttgg agataaatta aactttctat 50280 agttctgttt ctctaaaatt tgtttttctc tttaccttat agtcccgcag tattgatgag 50340 gagaccatta agacttaata tttttttgac acaatcttat atctcttcct ccaaccccta 50400 aaaagtgact gaggataggt acatcaagcc attgctttgt tactccccag gttttagtgc 50460 cagaccctga atggaagtgt caagcctttg gcctgtctga aaggtcattc ctgtgagcat 50520 atcatctccc ttccagctta cctctgtggc cattgcaaaa ggatttaaaa ataatttttg 50580 tgccatttga atggcacaag accagacagt gtatgtgggg gagtgtttct caaatcaaac 50640 tggaaactct ttaatttgta agaaccatta agcagagaga gaaaaaagaa aggaaaagaa 50700 aaaagatcct acagagaaca ccctgttcag tttgggaaca ggctacagct ttggattttt 50760 caaggcctag cattcccatc attctaaatt ttacttagct aatacaatag tagttgccag 50820 agctgatgac atagtatttt gtcatgcttg gctccgttca agcattttag ttttttagcc 50880 attaccatgg ctagacccag tcaaaagaat tttcattgtt taagattccc attatcctag 50940 tttttactag tagccagcca aagaaaagaa aaaggaggtc agaatttcgg tatttacata 51000 gaaatttaag gggaaaaggc caggcatgtt tttaaagtgt ggaaattaag aactattcat 51060 tatcccactg attgtgtgga tgtgtttttt aaagttttgt tactgtcttg agagagagaa 51120 tattgagata ggacataatg ttggtttaag ggaatgaggg tactttctgt aggtgaggtg 51180 ccaagccatg tcatcagaaa tgttagtcac atgactttct aagcacacct taaatgtttt 51240 accgtgtatg tttttgtaaa gttttaaatt tttaactggg aaaaacagac ctgtatatta 51300 agttttatat atatatataa atttaaaatt acatatatat gtttatatat gtaactttta 51360 tatgggagag atatatattt ctatatcctc tataaaaaaa catatctata tatgaaaatt 51420 atgtacgtaa atgttaattt ataattaatt atataaatat taacataatt acattatata 51480 tatagaaaac ctagtgtaca gatctgtata taaattaaaa atgtatgtgt tatatatagt 51540 tacatcatat aatacatata attgatatat ataatgataa atactttatt gaaggatgaa 51600 aaaatttcca tgctgtctca taaaataaga tggttgacat atgctaaact agatagattc 51660 tcctgtttca tactaaagca gaatgttgta aaatattaaa tccaaatgag atgtctcaga 51720 ttaaggccat ttcaacagga atgctgagac tttaaaaaaa aaaaaagtct gaggctgggc 51780 gtggtggctc atgcctgtaa tcccagcact ttgggaagct gaagcaggtg gatcacttga 51840 ggccaggagt ttgagaccag cctggccaat gtggtgaaat cccgcctcta ctaaaataca 51900 aaaaaaatac atgggtgtgg tgacgcatgc ctataattcc agctacttgg gaggctgagg 51960 caggagaatc acttgaacct gggaggtgga gattgcagta agccccacca ctgcactcca 52020 gcctgggcga agagcaaaac cctgtctcaa aaaaaaaaaa agcctgaatt atatcagcaa 52080 atgaaaactg taatgttgtt ctctgtttca gaggcccttg aatgaatagc actaaaaata 52140 ttttttaaaa aatgaagaaa atgaaaattg taatgttcct tatttaaaag gcccttgaat 52200 gagtagcatc aaaaatattt ttaaatggga ggccagggtg ggaggtttgt ttggcaccag 52260 gagatcaaga ccagcttggg taacatagca agacctttgt ctctaccaaa aaaaaaaaat 52320 tgggtgtggt ggtgccacct gtattcctag ctactgggaa cactgatgca ggaggatccc 52380 tgggactcta gagtccagag tgagaccctg tctctaaaac aaacaaacaa acaaaaactg 52440 tatttatgta aaagtaatac ttgtttttta aattttattt atttttaatt gataaaaatt 52500 gtatgtatgt ttatgtgatg tatatattgt ggaatggtta aatcaggcta attaactcag 52560 attttttgtg tgtgtgggga gaatatctaa aatccctctc cttagcagtt tccaaatgaa 52620 atgaaagaat aaaagtgatt tatttttttg agacagcatc tcaccctgtt tctcaggctg 52680 gaatgcagtg gcacgatctt ggcttacttg atcctcgact tccctggcat ccggtgatcc 52740 tcccacttca ctctcctaat tagctaggac tacaggcatg cgccaccatg actggctaat 52800 ttttgtattt cttgtatagg caaggttttg ccatgttgcc caggctggtt tcaagctcct 52860 gggctcaaac gatccacctg cctcagcctc ctgaagtgct gggattacaa gtgtgagcca 52920 ccacacctgg cgaaaagtgt tattttttta aatgacaaat ttaagtcaaa gagattgaat 52980 gttcacttct ggtactttgt atataagaga aacattccat taaataattt tttaaacatt 53040 tctaaaatta catattttgt cattaaatgt ttaaacaatc agtataattt cattgataca 53100 gtgtttgtta ttttgtcggt gtttaagatt gataattggg gttagtttta attcagaatg 53160 ttattctatt taatgtcaca cttcatgtct ttttattttg tatatctatt aatgaattat 53220 tttagctata gttattactg ttttagagat gaggtcttct atgttgccca gggtagactt 53280 gaactcctgg gcttcagcaa tcccctcctc aacctccgga gcacatgaga ttagagacgt 53340 gtgccactgt atctggcctg ctgtagttat ttttaattct tttgtctttc aacttttata 53400 ctagagttag aaatgattta caaaccctat tgcagtttta gagcgttatg aatttgacta 53460 tatatttctt ataacaactt aacttcagtt gcttacaaaa actacagagt tttactcccc 53520 cgtccacatt ttatactatt gatgtcacac tttacatctt tttattttgt gaatccatta 53580 atgatacttc tggtagtttt tacactccac tattcagttg tcagacacca ttcagttgtt 53640 agattgttat gagctaaaag caacttaatg ggtatttttc aaaaatcatt tatgtcaatt 53700 gctaatggac ttcttttcta tgccatgatc atgctttttt tatttttgag acggagtttc 53760 actcttgttg cctgggctgg agtgcaatgg cgcggcctca gctcactgca acctccgcct 53820 cctgggttca agcgattctc ctgcctcagc tgggattaca ggcatgtgcc accgtgccgg 53880 ctaattttgt atttttagta gagacagggt ttcaccatgt tggccaggct ggtctcgaac 53940 tcctgacctc agttgatctg cccaccttgg cctcccaaag tgctgggatt acagacgtga 54000 gccactgcgc ctggcctgat catgctttta aggtggttga gtaagtacta gttgctgggg 54060 ctttacttag tgccctccta ctcaaatgtg ttagaacata gttaagaagg ctgtagtgtt 54120 caaaaggagt aaaaagcagt gcagtgtttg cagtaatatc tgcttctcaa tttaggactg 54180 atgcttatta tggcttaaat gtttttgtag taaaatttgt attcaaaaaa tatatttttt 54240 tttctttttg cgacagagtc ttgctttgtc acccaggctg gagtgtggtg gtatgatcat 54300 ggctgactgc agccctgacc ttccgggctc aagtgatctt tccacctcag cctcccaatt 54360 acttgggacc accagcatgc ttggccgatt tttttttttt tttttttttt gtagaagcaa 54420 ggtttcccta tgttgccaag gctggtcttg aactttaggg ctcatgtgat actcctgcct 54480 cggcctccca aagtgttagg attacaagcc tgagccacca tggccggcca aaatattttc 54540 actataacaa atatcatatc tgtatatact cagttttaat actaactcaa agtagaaaca 54600 taaagctgaa tgactatttt attttcagat tctctccatt gagtttcctt ctccgtcttg 54660 tgtgatctct gaacttttct ccatctttgc cacttcttgt ctagcatttt ttttttatca 54720 gcagtttcat tcagattttt tttttagttc tttcaacggt ggagtggaag taggcagcag 54780 gacagaagaa cttgaagcag agcacactgg agaggagaaa ttaacaaagc ctttatgaat 54840 aaaacaaccc cccaatatca gtctgtgtgc attatgagca taattgtact ttcatctcat 54900 ctgtaatgtt catgactttt ctagaaaatt atactttaac atgagaaaag aaaaagaacc 54960 agctaattca tagggatgga ggacacagca tagtcaaagc aagaatgaaa ctctctttag 55020 tgccacctcc agtgcagaat aagtaacatt cagcagaggc aggtttcatt tgataatgga 55080 ttcctataat aaactgcgct cagaatttgt gcaggtttta aaatcccgta ttccaaaccc 55140 acttccttag cccccaagtt agaaaacagc ttcagtaaag aaaattgtac gatgatataa 55200 ctttaccaaa aaataatttc tttccatgaa gatgatatat tattgttgac ttctaattca 55260 atcaaatata aacaattgct aaatggcttt tcagttgact cctttcttgg ttaaggagaa 55320 gataggaaaa aatgaaggga tcagaagtca taggatacat taattttttt tatctctgaa 55380 taaacaggtt gcctacttaa aaatctatca gtttaaaagt gttggtctct tctctctctt 55440 ttcagaaagt gccaggaaaa aacaagaagg tatcgttggc agttctcggg tgtgttttgc 55500 acagcacacc ccatctcttc cagcagaaag tcctcggcca ttgaagcttc gaagcattct 55560 tgacatgagc ccttttacag tgacagacca caccccaatg gagatcgtgg tggatatttt 55620 ccgaaagctg ggactgaggc agtgccttgt aactcacaat gggtaagtct ggtaccacag 55680 gaatcagttc acttgctaga atataggatc ctttttagtg gaatctatat agttattagg 55740 ggagcatgtg agtcagctcc caggtgggaa agtctgtcct atggtatagt cacaaatata 55800 ggatcagtca atcaaatttc acatttacta aggaataaga aagatgtcat ctgcctgctc 55860 tttgccaaac agtgacattt gtaaataata cctcaaagtt ggaaaagagg tgctgaaaga 55920 tctccagcat gaaagcatgt tgagcttaga gtgcttcttt tcctagggaa gagtggacct 55980 aacctgcatg gagcactgca aaaacctgtt ttatttttgt aaatgtttca tttttagtat 56040 ataaatttct agtacaataa taagtttcta gatattttgc tatttactct ttcagccaat 56100 atttgattta tcatgtaatg aaggaaagaa tatatactta aatgaaattt gtaaatgagc 56160 taaaaatctc ctttaacaaa tgctttgttt ccttttgtct acctttctct atacacaaat 56220 cttttatatt tatataactg ctaaggacaa ataaatactc atgtatttaa aatgtataca 56280 ttgataattt atttttccac cttttacaca tgaactgcca gtgtttctcc attgacagga 56340 atataggaaa gaaacagatg tcacgggggt tgtggagacc ttaatgcaca gaattgattt 56400 agcaaataca ctacttcgtc accactgctc tcttttcctg gacctgggat ctgtttctcc 56460 acacttcttt ctttaggacc cttcatttcc actatatatt ctttcttgtt gaacttaaga 56520 atgttgtttt atccgaaggc aaataccaaa aaacagaggg tattcttgga ttatgcataa 56580 actggatggc taatcctgaa cagcgtaaag ctggttgaaa ttctaaacag agaatcatag 56640 cagttttttg ttgttttttt tttttaacat gttgtagaaa acacattggt gacagaatac 56700 atgactcctg tccagagaaa ggagagaaaa agaacagaaa ggaaggaaat ttgtttattg 56760 aacaccttca tattttctca tttaactttg caggacctct gcaaagtagg tagttatatc 56820 cctactttac agatgtagta attaaagctc aggaagcttt aataatttgc ccaaagtcat 56880 gtggtgaaca agtcatggtt caaggaatca gactgtcttt cctactttaa aacccagcct 56940 cttgctacta ttttgcactg taagtgactg atagaaatcc tctttctttg tgatttctta 57000 aactactaaa acattttctt ggccaatata ttagattgag ttaagaatag aaatatgaaa 57060 ctagagaatt agatctatgt ttagtgtttt tcactgcgct aattaaaata actctttagg 57120 aatatgaagt aaatcattaa agagataaag cccttaaagg cagggagttt agaattatta 57180 aattctaata atttagatac tgattggaga agagatgtat tcataagtta ttattgttac 57240 tatttgtctt tgtgtaatat tgtttgatta aatgatggca ccgacttcat taagtttaaa 57300 aactcagtac tagttaaatg gggcaacttt tcataaagct ttgctagtcc ttgagccctt 57360 ttatttgtta aatggctcaa ctggaaccta agctgagttg ttacaaacta ttatttgctt 57420 caagttgttt tctgttcctg gcatggcttt ttcttttgtg tactgacaaa tataaatgtt 57480 attctgttga gttatggtta actatgaaca cagaactgtt agggattaat tttcatattt 57540 cagtttgttg attaattccc aggtatttgg cagcatagat attagaaagg aaaatattta 57600 aaagaaagtg taaaaataac gaagtgtata gagcgagggg tggatagcta attaaaattt 57660 tgtctggtcc tgcctgttca tatgaaaaaa ggggttggac tttcttctaa gggaatatat 57720 taaattgctt tcatcatatt ttccttattt ctgtctgtca aggaaaataa attgatacat 57780 atatggggag aaaagagatc atttagggaa gtggctcatg ggactttttg ttttgtttga 57840 agtgtattag gaagtcgggt gttttttttc tcacttaaat tatttaaaac ccagaaaaga 57900 aatgatatct tctggttttt aaaggagacc atgaagttct gcatagctat cattgatgtg 57960 tagttcatac tgcattttta gaagtggaaa atagttattt ggaggaagat aacaaatctg 58020 gaaccttagg tgcaaggaga aaaagaatag atgaaaggga aagatgtttg taaattataa 58080 aaatttcaat tagctattgg ttttctgcac tttatatttt aactgcagaa tttttcaaaa 58140 tcagttaatc ttggtggaat tagcaggatg ttaataggag tgactcagaa aaaaacattt 58200 tgtgactgtc taagtttgga aagtattgga ttaaatacaa ttgaggtttc tttactatgg 58260 aactcctcag aacttataat atgttgatat tctttgattc ccagatgagg ggatgggtaa 58320 taggatacat ggttttccag acttgtttga aaatgcaact atttttgggt tgcagggaag 58380 gatatagtag aactcatggg aactggtgtt tcttggaaca tgctttggaa atgctgggtt 58440 atgccctgtt aactcttaca tcattagttt ttagcccaaa aggaaacagc aaataatgtt 58500 ttatatgagc cacattttgc gttgattttc cttccactct gtaaaattac taaagcagca 58560 ctctgacttt attatgctca aatcgctctt ctccattaat gtgtgtttct ccatctttta 58620 gggtttttac tttataaata cagagattac tgtgtaaaat tctaaatttg ccactgggtc 58680 gttatacatt tgtaaccttc ctcacagtat attttgtgat ttggcagagt ttaccaatat 58740 agatgatact aactgaaatt aatcattctg tataattgga tagaaaagca tgagtaagaa 58800 ttcaattggt attatattta attaattgcc aagattttca catttcctga ctacaacaat 58860 aaaatcaaat gaattgatgg cttaaaaaaa agaaatctca aatgtttagt caatgaagaa 58920 catctattga atgagtgaat gttcattata tatagtgcat tttctgagct tttttggagg 58980 gggaagttgc tcccatgctc tgagaacttt taaggatcga tacattattt ttaacataat 59040 aatgagaaaa catgagcaga gaacccattt ctgtcattcc cattctctat cctcctgctc 59100 ccccacctcc caccccagcc atcaagctaa gtaactattt tacacctgga cgtagctata 59160 ggaacaggct actttgaagt ctcctagtga catccttcaa gtctgaatgt tcaaaggcag 59220 tttaacaggg aggttgactt aatgagatca tcaaggaaat gtccagtcat cctgaagggt 59280 attttggatg ggcttccaga atttaaagat taaagttttt ttaaggtttt tttattttca 59340 ctgtttatat tgccacatta atttccatta taaaaccagt aaccatagtt ttgttttaat 59400 tagcaatcta attattttca tgtatcctca ttatgagaat ttatgtccat cactttgctt 59460 gatgtgataa cagtgacatg ctaaatgaga aacaattgtt atttagaaaa aaatgcacaa 59520 agtgaaagtc cttttaatcc ctaatcataa atacatttta ttagcttact ttaagaagtg 59580 gcagtcacag ctcctgaaca ttagggagtg tttcttttgg tcagcattat ttatttagtg 59640 cacattgcct ttaattttaa tttgaaatta tagtaaaatc cacgggagtt tttaagtctc 59700 ctcacagcct tttgctacct tttcaccaag gtagatccag atgataactg ctgtgttgtg 59760 acatcataga aattagaaaa atattttcct ctgaggaaag aacattgtaa atgaaactct 59820 acatatcaga ggtctatagc tatgtatcaa tattaagttt cttttgtact ttgctttgta 59880 gtcatcttca ttccaaactt tcataattat tatttttact ttaaaaagaa aaataaccca 59940 ccaatattga agattagtat tgtgtcactt ttgaaagtca gtagaattta tgcaaaagga 60000 acctggaact ttaaatcatt ttgtttttat tttctaaagt tcatgagact cattcttatg 60060 gttcatgttt ttattttttc tctcattctt tatcattatg attggaaact cttttaattt 60120 aatttctcac acagttatta gcataataat ctgtttcagg attgtcttgg ggatcatcac 60180 aaagaagaac atattagagc atctcgagca actaaagcag cacgtcgaac ccttggtgat 60240 tagatatatc agatctcctc attagacacc ttagaagtca ggaagcatga aacttgtgaa 60300 ctgttgagtt ctgtctttcc cagatatctg ctgaacaaaa atatcctact atgctgccaa 60360 ttacatttgt atctgataaa atgtgtctgt aagataaatt tagatatgtg taaaatccca 60420 tttatagaaa gtaagcaaaa gttaacatct ctcatcaaat cattcattac aatttcagaa 60480 ctgtaaacag tttggtagtg gaataagtga atattattgg acattcttaa agtgaatatg 60540 gcaaatctgt ctacctcagt ggatacaccg gtctcagaag acacctgact ggttaaaaat 60600 gtctgaccca tccccgcaag cccttttttt tttttttaaa tgtttcccga tcttgtggta 60660 gtcttatggt aaatctaagc tcctaaagga ttttaaagga gcttagcaat tagaactgct 60720 tacagttaaa tggatttttt aatgggcaca ctaactagag tgtaatgtgt atattatttg 60780 tgatcatagc attagttctt tttctgctat accctgcata tcttcaaagt cacagtgtgt 60840 gtcctgccat ctcattagtg aattgtacct agattatgtg tgtgcccctt ttgtatgatg 60900 tttctggaac gctataagca gcttttagag tcaaatgcat tcattttaac tggctttatg 60960 tcctagtggt ttcatgacta caaatttgaa ttatcttact gcataacata aaaaatgtct 61020 ggctttagca attaatgccc gaaattattt tgccctgcaa ttgtcatacc tgtatgaaac 61080 ctgtcccagt ttgcttaagt gcacaactga ttatgtattc ctgtgtgtat gctaatattt 61140 cacaagtgtt tcatgcatcc ttttttaaaa aactactaac cagaatatta tcgtagctac 61200 tcattcattc tgctttctgc ttcacctata ataatctttt aggactgcct tctgattttt 61260 cacctatctt ttaatgtaag cattaacaac taagactttc ataaaagcac tgtatcttaa 61320 ctttcctggc ctaaatcaaa aaaaggaaaa cattgataag tgtcctagaa acttggattc 61380 ttttatagat ttgttcttgg ggctctgatg tttgggattg acgttctgtg ctgaccattt 61440 tatatgcatt ttatcttaat agtatgtgct ttcatgaaga ttctgataca agtgggcaat 61500 ccttaaatta tctttgaaaa attggttaat tttggttaaa aaagggaaag tggctgggtg 61560 cagtggctca cgcctgtaat ccccagcact ttgggaggcc gggacgggtg gatcacaagg 61620 tcaggagttg aagcccattc tggccaacat ggtgaaaccc tgtctctact gaaaataatt 61680 ggggcatggt ggcacatgcc tgtaatccca gctacttggg aagctgaggc aggagaattg 61740 cttgaaccgg ggacccagga ggcggaggtt gcagtgagct gagatcgcgc cactgcactc 61800 cagcctgggc tacagagcga gactctgtct caaaaaataa ataaataaat aaatgaaaaa 61860 gagaaaatat tgagaggatt tggtcatcat tttactgctc tcttcatgtg atggaaatca 61920 attttccttc tcaaatggga tcagtatcat ttcctagtca tacatccatc cagtttttgt 61980 tacttttttg ttggcataca ttaatcaaaa tagctctgct tcattgaggc atgcagtcct 62040 cagactctcg gtggaaaggc tgtcatacta ttagtgacca tagtaacttt ttataccaaa 62100 ggatggttgc tggataattt taatatcttt accaataaag tactttttgg aaatacaaaa 62160 tcaggctgct tgctttgctc tattcctgtc aacaaaaagg atttagctat agatttagct 62220 tctcctttta ttttcccttt tatttcatag gagtcttctg tttattcctt tcaggcgcct 62280 ccttggcatt ataacaaaaa aagatatcct ccggcatatg gcccagacgg caaaccaaga 62340 ccccgcttca ataatgttca actgaatctc acagatgagg agagagaaga aacggaagag 62400 gaagtttatt tgttgaatag cacaactctt taacctgagg gagtcatcta cttttttttc 62460 ctcctttaca aaaaaagaaa ggaaatataa aagccgggtt tttgcaacat ggtttgcaaa 62520 taatgctggt ggaatggagg agttgtttgg ggagggaaag gagagagaag gaaaggagtg 62580 aggtatttcc cgtctaacag aaagcagcgt atcaactcct attgttctgc actggatgca 62640 ttcagctgag gatgtgcctg atagtgcagg cttgcgcctc aacagagatg acagcagagt 62700 cctcgagcac ctggcctgtt gctccaacat tgcaaagaca cattatcagt ccctatttct 62760 agagggatta ctttgaattg agccatctat aaaactgcaa ggtcttgccc ttttttttaa 62820 tcaaaactgt tctgtttaat tcatgaattg tatagttaag cattaccttt ctacattcca 62880 gaagagcctt tatttctctc tctctctctc tctctctctc tctctctact gagctgtaac 62940 aaagcctctt taaatcggtg tatccttttg aagcagtcct ttctcatatt gagatgtact 63000 gtgattttac tgaggtttca tcacaagaag ggagtgtttc ttgtgccatt aaccatgtag 63060 tttgtaccat cactaaatgc ttggaacagt acacatgcac cacaacaaag gctcatcaaa 63120 caggtaaagt ctcgaaggaa gcgagaacga aatctctcat tgtgtgccgt gtggctcaaa 63180 accgaaaaca atgaagcttg gttttaaagg ataaagtttt cttttttgtt ttcctctcag 63240 actttatgga taatgtgacc gggtcttatg caaattttct atttctaaaa ctactactat 63300 gatatacaag tgctgttgag cataattaaa taaaatgctg ctgctttgac agtaaagaga 63360 aggaagtatt ctgattagct gtatctggta ttaattgcat gttaaaacac tggaattttt 63420 aaaattgaaa ttagatcagt cattcttttc ttttctcaag atatctcatg gctgacactg 63480 aagaagaaat gtaattcata acttgcacta aatgtatatt ttttttctta aaaatttacc 63540 attcttattt atatttttat ggattaaaat ttataaaata cagatcagtt aatattgcac 63600 ttaagtaatt ttaccttttt aatgtgattt ttatagaata attcagactt acaaatacag 63660 agatatgaac aaagtttaca gtgggaacaa aggtttaaaa aaaggttgtg gttctctctc 63720 tgtgatccag tgtgcacata aacctttctc tgatctttca ctgccatcct ctggattatg 63780 tcttctgacc tgtccatttt gacccattaa ctggaaagtt gaaaaactac attaactgga 63840 aagttgaaaa actacattac tttggagaat aaaaccgaaa gttcgtgtat accttcttaa 63900 aaaaaaaatc aaaccaaaaa tgtgaaaaca atagaattgc aaagatagca gttaaaattt 63960 taatctgaaa ataacctttg aatctcgggc taggttatgt ccatatttga agtggtcagt 64020 gatggtttga acattttttg caggatgagt taaaatgcac tggattatat ttgggatttt 64080 tgtttttgga attgtctgtt ttaatcacag ccttaattca caattggcaa aggcagttta 64140 ctcaaaggac tgggctaaat attctgtaat tatgcatttt tgataggaaa atgaaatttt 64200 tgcaaacaga cattttcttt ttttttggct ggagtgcagt ggggcatggt cttggctcac 64260 tgcagcgttg accacctggg ctcaagtgat actcccgcct cagccaccca agtagctggc 64320 actacgggca cacgccacca tgcccagcta atttttttgt atttttagta gagatggggt 64380 tttgccatgc tgcccaggct ggtctcaact cctcagctca agcaatctgc ctgcgtgagc 64440 ctcccaaagt ggtggaatta caggcgtggg ccactgcgcc tggcccagac agacattttc 64500 tgaaacacaa ctggcaatga gctgttttta cattttgaaa gtgattcttc acttcctagt 64560 tcttaattat agtataccta ttaagatctg taagatcctg aagacataag atcatgaagc 64620 catataagaa tgaggattga aagttgagca aaattttcgg gattttggga aacattctta 64680 gctgtgctat ctgcctaaaa ttattcctta ttacttctct cctttgacag acttcaagtt 64740 ttcttcatag ccctttcaaa gttttttgag ccatccagag taaaatcatt tctaaatgat 64800 agttctgtat atctccaact cgtcttaagt gtatttgcct gtgtgcaacg tattgctaga 64860 ctatgaactc ctcagcatgg ctgctggata acttaattgt cctgagttaa tagccttcaa 64920 aggacaaatc ggtttctttg cagatagctt cgtaaaactt cacatggagt ttattttatc 64980 atatttccct tttttatttc tgctcctcct ttaattgccc atcttgcttc agagactgac 65040 atttcagggt ggatattaat taaagcatta attttgtttt ttggtatatt tctatcccta 65100 gtatttctat cttactgcta aaatacagga aaagtgccgt atttttaatg catttagtgg 65160 ttttctttgg tgttatctgt tccatttttc tttttcatac attgaagtgt gtctcctttt 65220 caaccaaaat aatgaaatag tggagaccat gaaattgttg tgcctggcta attggcaaat 65280 taatttacca atataataag tgtagcgcct tgtttgaata ccctttttga gaaggtatga 65340 tgagaatggg caagggtgt 65359 4 765 PRT Human 4 Gly Thr His Tyr Thr Met Thr Asn Gly Gly Ser Ile Asn Ser Ser Thr 1 5 10 15 His Leu Leu Asp Leu Leu Asp Glu Pro Ile Pro Gly Val Gly Thr Tyr 20 25 30 Asp Asp Phe His Thr Ile Asp Trp Val Arg Glu Lys Cys Lys Asp Arg 35 40 45 Glu Arg His Arg Arg Ile Asn Ser Lys Lys Lys Glu Ser Ala Trp Glu 50 55 60 Met Thr Lys Ser Leu Tyr Asp Ala Trp Ser Gly Trp Leu Val Val Thr 65 70 75 80 Leu Thr Gly Leu Ala Ser Gly Ala Leu Ala Gly Leu Ile Asp Ile Ala 85 90 95 Ala Asp Trp Met Thr Asp Leu Lys Glu Gly Ile Cys Leu Ser Ala Leu 100 105 110 Trp Tyr Asn His Glu Gln Cys Cys Trp Gly Ser Asn Glu Thr Thr Phe 115 120 125 Glu Glu Arg Asp Lys Cys Pro Gln Trp Lys Thr Trp Ala Glu Leu Ile 130 135 140 Ile Gly Gln Ala Glu Gly Pro Gly Ser Tyr Ile Met Asn Tyr Ile Met 145 150 155 160 Tyr Ile Phe Trp Ala Leu Ser Phe Ala Phe Leu Ala Val Ser Leu Val 165 170 175 Lys Val Phe Ala Pro Tyr Ala Cys Gly Ser Gly Ile Pro Glu Ile Lys 180 185 190 Thr Ile Leu Ser Gly Phe Ile Ile Arg Gly Tyr Leu Gly Lys Trp Thr 195 200 205 Leu Met Ile Lys Thr Ile Thr Leu Val Leu Ala Val Ala Ser Gly Leu 210 215 220 Ser Leu Gly Lys Glu Gly Pro Leu Val His Val Ala Cys Cys Cys Gly 225 230 235 240 Asn Ile Phe Ser Tyr Leu Phe Pro Lys Tyr Ser Thr Asn Glu Ala Lys 245 250 255 Lys Arg Glu Val Leu Ser Ala Ala Ser Ala Ala Gly Val Ser Val Ala 260 265 270 Phe Gly Ala Pro Ile Gly Gly Val Leu Phe Ser Leu Glu Glu Val Ser 275 280 285 Tyr Tyr Phe Pro Leu Lys Thr Leu Trp Arg Ser Phe Phe Ala Ala Leu 290 295 300 Val Ala Ala Phe Val Leu Arg Ser Ile Asn Pro Phe Gly Asn Ser Arg 305 310 315 320 Leu Val Leu Phe Tyr Val Glu Tyr His Thr Pro Trp Tyr Leu Phe Glu 325 330 335 Leu Phe Pro Phe Ile Leu Leu Gly Val Phe Gly Gly Leu Trp Gly Ala 340 345 350 Phe Phe Ile Arg Ala Asn Ile Ala Trp Cys Arg Arg Arg Lys Ser Thr 355 360 365 Lys Phe Gly Lys Tyr Pro Val Leu Glu Val Ile Ile Val Ala Ala Ile 370 375 380 Thr Ala Val Ile Ala Phe Pro Asn Pro Tyr Thr Arg Leu Asn Thr Ser 385 390 395 400 Glu Leu Ile Lys Glu Leu Phe Thr Asp Cys Gly Pro Leu Glu Ser Ser 405 410 415 Ser Leu Cys Asp Tyr Arg Asn Asp Met Asn Ala Ser Lys Ile Val Asp 420 425 430 Asp Ile Pro Asp Arg Pro Ala Gly Ile Gly Val Tyr Ser Ala Ile Trp 435 440 445 Gln Leu Cys Leu Ala Leu Ile Phe Lys Ile Ile Met Thr Val Phe Thr 450 455 460 Phe Gly Ile Lys Val Pro Ser Gly Leu Phe Ile Pro Ser Met Ala Ile 465 470 475 480 Gly Ala Ile Ala Gly Arg Ile Val Gly Ile Ala Val Glu Gln Leu Ala 485 490 495 Tyr Tyr His His Asp Trp Phe Ile Phe Lys Glu Trp Cys Glu Val Gly 500 505 510 Ala Asp Cys Ile Thr Pro Gly Leu Tyr Ala Met Val Gly Ala Ala Ala 515 520 525 Cys Leu Gly Gly Val Thr Arg Met Thr Val Ser Leu Val Val Ile Val 530 535 540 Phe Glu Leu Thr Gly Gly Leu Glu Tyr Ile Val Pro Leu Met Ala Ala 545 550 555 560 Val Met Thr Ser Lys Trp Val Gly Asp Ala Phe Gly Arg Glu Gly Ile 565 570 575 Tyr Glu Ala His Ile Arg Leu Asn Gly Tyr Pro Phe Leu Asp Ala Lys 580 585 590 Glu Glu Phe Thr His Thr Thr Leu Ala Ala Asp Val Met Arg Pro Arg 595 600 605 Arg Asn Asp Pro Pro Leu Ala Val Leu Thr Gln Asp Asn Met Thr Val 610 615 620 Asp Asp Ile Glu Asn Met Ile Asn Glu Thr Ser Tyr Asn Gly Phe Pro 625 630 635 640 Val Ile Met Ser Lys Glu Ser Gln Arg Leu Val Gly Phe Ala Leu Arg 645 650 655 Arg Asp Leu Thr Ile Ala Ile Glu Ser Ala Arg Lys Lys Gln Glu Gly 660 665 670 Ile Val Gly Ser Ser Arg Val Cys Phe Ala Gln His Thr Pro Ser Leu 675 680 685 Pro Ala Glu Ser Pro Arg Pro Leu Lys Leu Arg Ser Ile Leu Asp Met 690 695 700 Ser Pro Phe Thr Val Thr Asp His Thr Pro Met Glu Ile Val Val Asp 705 710 715 720 Ile Phe Arg Lys Leu Gly Leu Arg Gln Cys Leu Val Thr His Asn Gly 725 730 735 Arg Leu Leu Gly Ile Ile Thr Lys Lys Asp Ile Leu Arg His Met Ala 740 745 750 Gln Thr Ala Asn Gln Asp Pro Ala Ser Ile Met Phe Asn 755 760 765 5 767 PRT Human 5 Gly Thr His Tyr Thr Met Thr Asn Gly Gly Ser Ile Asn Ser Ser Thr 1 5 10 15 His Leu Leu Asp Leu Leu Asp Glu Pro Ile Pro Gly Val Gly Thr Tyr 20 25 30 Asp Asp Phe His Thr Ile Asp Trp Val Arg Glu Lys Cys Lys Asp Arg 35 40 45 Glu Arg His Arg Arg Ile Asn Ser Lys Lys Lys Glu Ser Ala Trp Glu 50 55 60 Met Thr Lys Ser Leu Tyr Asp Ala Trp Ser Gly Trp Leu Val Val Thr 65 70 75 80 Leu Thr Gly Leu Ala Ser Gly Ala Leu Ala Gly Leu Ile Asp Ile Ala 85 90 95 Ala Asp Trp Met Thr Asp Leu Lys Glu Gly Ile Cys Leu Ser Ala Leu 100 105 110 Trp Tyr Asn His Glu Gln Cys Cys Trp Gly Ser Asn Glu Thr Thr Phe 115 120 125 Glu Glu Arg Asp Lys Cys Pro Gln Trp Lys Thr Trp Ala Glu Leu Ile 130 135 140 Ile Gly Gln Ala Glu Gly Pro Gly Ser Tyr Ile Met Asn Tyr Ile Met 145 150 155 160 Tyr Ile Phe Trp Ala Leu Ser Phe Ala Phe Leu Ala Val Ser Leu Val 165 170 175 Lys Val Phe Ala Pro Tyr Ala Cys Gly Ser Gly Ile Pro Glu Ile Lys 180 185 190 Thr Ile Leu Ser Gly Phe Ile Ile Arg Gly Tyr Leu Gly Lys Trp Thr 195 200 205 Leu Met Ile Lys Thr Ile Thr Leu Val Leu Ala Val Ala Ser Gly Leu 210 215 220 Ser Leu Gly Lys Glu Gly Pro Leu Val His Val Ala Cys Cys Cys Gly 225 230 235 240 Asn Ile Phe Ser Tyr Leu Phe Pro Lys Tyr Ser Thr Asn Glu Ala Lys 245 250 255 Lys Arg Glu Val Leu Ser Ala Ala Ser Ala Ala Gly Val Ser Val Ala 260 265 270 Phe Gly Ala Pro Ile Gly Gly Val Leu Phe Ser Leu Glu Glu Val Ser 275 280 285 Tyr Tyr Phe Pro Leu Lys Thr Leu Trp Arg Ser Phe Phe Ala Ala Leu 290 295 300 Val Ala Ala Phe Val Leu Arg Ser Ile Asn Pro Phe Gly Asn Ser Arg 305 310 315 320 Leu Val Leu Phe Tyr Val Glu Tyr His Thr Pro Trp Tyr Leu Phe Glu 325 330 335 Leu Phe Pro Phe Ile Leu Leu Gly Val Phe Gly Gly Leu Trp Gly Ala 340 345 350 Phe Phe Ile Arg Ala Asn Ile Ala Trp Cys Arg Arg Arg Lys Ser Thr 355 360 365 Lys Phe Gly Lys Tyr Pro Val Leu Glu Val Ile Ile Val Ala Ala Ile 370 375 380 Thr Ala Val Ile Ala Phe Pro Asn Pro Tyr Thr Arg Leu Asn Thr Ser 385 390 395 400 Glu Leu Ile Lys Glu Leu Phe Thr Asp Cys Gly Pro Leu Glu Ser Ser 405 410 415 Ser Leu Cys Asp Tyr Arg Asn Asp Met Asn Ala Ser Lys Ile Val Asp 420 425 430 Asp Ile Pro Asp Arg Pro Ala Gly Ile Gly Val Tyr Ser Ala Ile Trp 435 440 445 Gln Leu Cys Leu Ala Leu Ile Phe Lys Ile Ile Met Thr Val Phe Thr 450 455 460 Phe Gly Ile Lys Val Pro Ser Gly Leu Phe Ile Pro Ser Met Ala Ile 465 470 475 480 Gly Ala Ile Ala Gly Arg Ile Val Gly Ile Ala Val Glu Gln Leu Ala 485 490 495 Tyr Tyr His His Asp Trp Phe Ile Phe Lys Glu Trp Cys Glu Val Gly 500 505 510 Ala Asp Cys Ile Thr Pro Gly Leu Tyr Ala Met Val Gly Ala Ala Ala 515 520 525 Cys Leu Gly Gly Val Thr Arg Met Thr Val Ser Leu Val Val Ile Val 530 535 540 Phe Glu Leu Thr Gly Gly Leu Glu Tyr Ile Val Pro Leu Met Ala Ala 545 550 555 560 Val Met Thr Ser Lys Trp Val Gly Asp Ala Phe Gly Arg Glu Gly Ile 565 570 575 Tyr Glu Ala His Ile Arg Leu Asn Gly Tyr Pro Phe Leu Asp Ala Lys 580 585 590 Glu Glu Phe Glu Phe Thr His Thr Thr Leu Ala Ala Asp Val Met Arg 595 600 605 Pro Arg Arg Asn Asp Pro Pro Leu Ala Val Leu Thr Gln Asp Asn Met 610 615 620 Thr Val Asp Asp Ile Glu Asn Met Ile Asn Glu Thr Ser Tyr Asn Gly 625 630 635 640 Phe Pro Val Ile Met Ser Lys Glu Ser Gln Arg Leu Val Gly Phe Ala 645 650 655 Leu Arg Arg Asp Leu Thr Ile Ala Ile Glu Ser Ala Arg Lys Lys Gln 660 665 670 Glu Gly Ile Val Gly Ser Ser Arg Val Cys Phe Ala Gln His Thr Pro 675 680 685 Ser Leu Pro Ala Glu Ser Pro Arg Pro Leu Lys Leu Arg Ser Ile Leu 690 695 700 Asp Met Ser Pro Phe Thr Val Thr Asp His Thr Pro Met Glu Ile Val 705 710 715 720 Val Asp Ile Phe Arg Lys Leu Gly Leu Arg Gln Cys Leu Val Thr His 725 730 735 Asn Gly Arg Leu Leu Gly Ile Ile Thr Lys Lys Asp Ile Leu Arg His 740 745 750 Met Ala Gln Thr Ala Asn Gln Asp Pro Ala Ser Ile Met Phe Asn 755 760 765 6 60 PRT Xenopus laevis 6 Met Asp Ile Ser Ser Asp Pro Tyr Leu Pro Tyr Asp Gly Gly Gly Asp 1 5 10 15 Asn Ile Pro Leu Arg Asp Leu His Lys Arg Gly Thr His Tyr Thr Val 20 25 30 Thr Asn Gly Gly Ala Ile Asn Ser Thr Thr His Leu Leu Asp Leu Leu 35 40 45 Asp Glu Pro Ile Pro Gly Val Gly Thr Tyr Asp Asp 50 55 60 

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acid molecule of claim
 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim
 5. 9. A host cell containing the vector of claim
 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.
 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.
 18. A method for treating a disease or condition mediated by a human transporter protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 20. An isolated human transporter peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:2.
 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:2.
 22. An isolated nucleic acid molecule encoding a human transporter peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
 3. 